Extensive Cargo Identification Reveals Distinct Biological Roles of the 12

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Extensive cargo identification reveals distinct biological roles of the 12 importin pathways Makoto Kimura1*, Yuriko Morinaka1, Kenichiro Imai2,3, Shingo Kose1, Paul Horton2,3, Naoko Imamoto1*

1Cellular Dynamics Laboratory, RIKEN, Wako, Japan; 2Artificial Intelligence Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan; 3Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan

Abstract Vast numbers of proteins are transported into and out of the nuclei by approximately 20 species of importin-b family nucleocytoplasmic transport receptors. However, the significance of the multiple parallel transport pathways that the receptors constitute is poorly understood because only limited numbers of cargo proteins have been reported. Here, we identified cargo proteins specific to the 12 species of human import receptors with a high-throughput method that employs stable isotope labeling with amino acids in cell culture, an in vitro reconstituted transport system, and quantitative mass spectrometry. The identified cargoes illuminated the manner of cargo allocation to the receptors. The redundancies of the receptors vary widely depending on the cargo protein. Cargoes of the same receptor are functionally related to one another, and the predominant protein groups in the cargo cohorts differ among the receptors. Thus, the receptors are linked to distinct biological processes by the nature of their cargoes. DOI: 10.7554/eLife.21184.001

*For correspondence: makimura@ riken.jp (MK); [email protected] Introduction (NI) In interphase cells, proteins and RNAs migrate into and out of the nuclei through the central chan- Competing interests: The nels of the nuclear pore complexes (NPC) embedded in the nuclear envelope. These nuclear pores authors declare that no are lined with FG-repeat domains that constitute a permeability barrier, and only macromolecules competing interests exist. that reversibly interact with the FG-repeats can permeate this barrier (Schmidt and Go¨rlich, 2016). One such group of proteins, the importin (Imp)-b family proteins, are nucleocytoplasmic transport Funding: See page 23 receptors (NTRs) that primarily carry nuclear proteins and small RNAs as their cargoes through the Received: 02 September 2016 nuclear pores, although non-importin family NTRs also act depending on the cargo and physiological Accepted: 17 January 2017 conditions (Kose et al., 2012; Lu et al., 2014; Weberruss et al., 2013). The human genome enco- Published: 24 January 2017 des 20 species of Imp-b family NTRs, of which 10 [Imp-b, transportin (Trn)-1, -2, -SR (-3), Imp-4, -5 Reviewing editor: Karsten Weis, (RanBP5), -7, -8, -9, and -11] are nuclear import receptors, 7 [exportin (Exp)-1 (CRM1), -2 (CAS/ ETH Zurich, Switzerland CSE1L), -5, -6, -7, -t, and RanBP17] are export receptors, 2 (Imp-13 and Exp-4) are bi-directional receptors, and the function of RanBP6 is undetermined (Kimura and Imamoto, 2014). These NTRs Copyright Kimura et al. This constitute multiple parallel transport pathways. The basic mechanism of directional transport was article is distributed under the elucidated in the early years (Go¨rlich and Kutay, 1999), but even today, the number of NTR-specific terms of the Creative Commons Attribution License, which cargoes that has been reported is surprisingly small, hindering a biological understanding of the permits unrestricted use and nucleocytoplasmic transport system. redistribution provided that the NTRs are thought to transport specific cohorts of cargoes by binding to specific sites on those original author and source are cargoes (Chook and Su¨el, 2011), but the consensus structures of the NTR-binding sites have been credited. established for only a few NTRs (Soniat and Chook, 2015) as follows: the classical nuclear

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 1 of 31 Tools and resources Biochemistry Cell Biology localization signal (cNLS) for the Imp-a family adapters, which connects Imp-b and cargoes (Lange et al., 2007); PY-NLS for Trn-1 and -2 (Lee et al., 2006; Su¨el et al., 2008); the nuclear export signal (NES) for Exp-1 (Hutten and Kehlenbach, 2007); the SR-rich domain that binds to Trn-SR (Kataoka et al., 1999; Maertens et al., 2014); and Lys-rich NLS (IK-NLS) for yeast Kap121p (Imp-5 homolog; Kobayashi and Matsuura, 2013; Kobayashi et al., 2015). The b-like importin-binding (BIB) domain is another NTR-binding site (Ja¨kel and Go¨rlich, 1998), but its consensus sequence and NTR specificity remain obscure. Among the NTRs, Imp-b exclusively uses one of the seven species of the Imp-a family of proteins as an adapter for cargo binding, and many Imp-a/b cargoes have been reported, although Imp-b also directly binds to cargoes (Goldfarb et al., 2004). Among the import receptors, Trn-1 and its closest homolog Trn-2 have the second-highest number of cargoes reported thus far, and the PY-NLS motif has been defined, although in some cases the motif is difficult to rec- ognize because of sequence diversity and structural disorder is another requisite (Soniat and Chook, 2015, 2016). For the cargoes of other NTRs, the consensus structures of NTR-binding sites have hardly been derived because only limited numbers of cargoes have been reported, including Imp-b- direct cargoes. There are many reports on the differential spatiotemporal expression of Imp-b family NTRs, including tissue specificities in humans (Quan et al., 2008), developmental or spermatogenic stage specificities in mice (Major et al., 2011; Quan et al., 2008), and tissue or response specificities in plants (Huang et al., 2010). Expression regulation is not only transcriptional but also miRNA-mediated (Li et al., 2013; Szczyrba et al., 2013) or locally translationally mediated (Perry and Fainzilber, 2009). Additionally, the NTRs are functionally regulated by protein modifications (Wang et al., 2009), inhibitory factors (Lieu et al., 2014), and specific anchorings (Makhnevych et al., 2003). These nucleocytoplasmic transport regulations must significantly influence cellular physiology, and their significance may be elucidated if the affected cargoes can be specified. Indeed, in previous studies, NTR regulations have been linked to cellular responses through the functions of specific cargoes. For example, in prostate cancer cells treated with a cinnamaldehyde derivative, the expression of Imp-7 and the transcription factor Egr1 are induced, and the Egr1 imported by Imp-7 activates apoptotic gene transcription (Kang et al., 2013). In another example, when the nuclear import of some ribosomal proteins (RPs) is inhibited by the repression of Imp-7 expression, other unassembled RPs restrain the negative regulator of p53 Mdm2 and thereby activate p53 to inhibit cell growth (Golomb et al., 2012). Additionally, the inhibition of Trn-2 by the caspase-generated HuR (ELAVL1) fragment is crucial for the cytoplasmic retention of full-length HuR, which induces myogenesis (Beauchamp et al., 2010) or staurosporine-induced apoptosis (von Roretz et al., 2011). In many other studies, mutations of particular NTR genes in model organisms, including yeast, flies, and plants, have resulted in defects in specific biological processes (Kimura and Imamoto, 2014). Thus, each NTR has its own inherent biological significance. However, the details of the molecular processes are largely uncharacterized because the responsible cargoes have not been identified. If we could identify more cargoes, further studies of cellular regulation by nucleocytoplasmic transport would be possible. We previously established a method for identifying the cargoes of a nuclear import receptor called SILAC-Tp (Kimura et al., 2013a, 2013b, 2014). SILAC-Tp employs stable isotope labeling with amino acids in cell culture (SILAC) (Ong et al., 2002), an in vitro reconstituted nuclear transport system (Adam et al., 1990), and quantitative mass spectrometry. A recent advancement of the Orbi- trap mass spectrometer drastically increased the identified and quantified protein numbers, and this advancement has been successfully applied to other cargo identification methods (Kırlı et al., 2015; Thakar et al., 2013). Here, we utilized this advancement for the SILAC-Tp method and identified import cargoes of all 12 NTRs, of which 10 are import and two are bi-directional receptors. Our results illustrate the basic framework and the biological significance of the nucleocytoplasmic transport pathways.

Results and discussion SILAC-Tp effectively identifies cargoes SILAC-Tp employs an in vitro nuclear transport system, and all 12 NTRs import their reported specific cargoes in this system (Figure 1—figure supplement 1C). The transport system consists of

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 2 of 31 Tools and resources Biochemistry Cell Biology permeabilized HeLa cells labeled with ‘heavy’ amino acids by SILAC, unlabeled HeLa nuclear extract depleted of Imp-b family NTRs and RCC1, unlabeled HeLa cytosolic extract depleted of Imp-b family NTRs, one species of recombinant NTR, p10/NTF2, and an ATP regeneration system. Unlabeled ‘light’ proteins in the nuclear extract are imported into the nuclei of the permeabilized cells. Simulta- neously, a control reaction without the NTR is performed. Next, the proteins are extracted from the nuclei and identified and quantified by LC-MS/MS. The recipient nuclei contain both the imported and endogenous proteins, and the ratio of the imported to the endogenous fraction of a protein is calculated as the unlabeled/labeled or light/heavy (L/H) ratio. The quotient of the L/H ratios with the

NTR (+NTR) and without it (control), that is, (L/H+NTR)/(L/HCtl), of a protein is defined as the +NTR/ Ctl value and is used as the index for cargo potentiality. In one run of SILAC-Tp (control or +NTR), approximately 2500 to 4000 proteins were identified, and the L/H ratios of 1700 to 3100 proteins were quantified. To calculate the +NTR/Ctl value, one

protein has to be quantified in both the control and +NTR reactions, and we discarded L/H+NTR val-

ues that lacked the counterpart L/HCtl values. We performed three replicates of SILAC-Tp for each of the 12 NTRs. In the three replicates, 1235 to 1671 proteins were assigned with +NTR/Ctl values three times, and 364 to 502 proteins were assigned only twice (Supplementary file 1). We did not consider proteins with single +NTR/Ctl values, although a protein with only a single but high +NTR/ Ctl value may still be a cargo (see below). To normalize the index values of the three replicates, the

Z-scores of the log2(+NTR/Ctl) were calculated within each replicate (Figure 1—figure supplement 2A and B). Ranking the proteins that have three +NTR/Ctl values by the median of the three Z-scores may reasonably sort the candidate cargoes. However, if the lower Z-score of a protein with only two +NTR/Ctl values is higher than the median Z-scores of those candidate cargoes, the protein may also be a candidate cargo. Thus, we ranked the proteins by the second (the lower of the two or the middle of the three) Z-scores, and termed the result the 2nd-Z-ranking (Supplementary file 1). To define the border that separates candidate cargoes from other proteins in the 2nd-Z-ranking, we first reviewed the distribution of reported Trn-1 cargoes in the Trn-1 2nd-Z-ranking because many Trn-1 cargoes have been reported. For an unbiased evaluation, we employed the lists of cargoes consolidated by other researchers (Chook and Su¨el, 2011). Twenty-seven reported cargoes were included in the 2nd-Z-ranking (totaling 1649 proteins; Supplementary file 1, Trn-1 ‘Report and feature’). We calculated the reported cargo rates (to serve as a proxy for precision), recall, and Fish- er’s exact test p-values for rank cutoffs in increments of 1%. Computing reported cargo rate requires deciding which candidate cargoes should be considered as false positives. Since a gold standard set of definitely non-cargo proteins is not available, it is not clear which previously unreported cargoes should be counted as false positives, and which, if any, should be discarded as unclear. Therefore, we estimated reported cargo rates in two ways: (i) treating all the 1622 proteins not reported as cargoes as negative examples (Figure 1—figure supplement 3A and Figure 1—source data 1A); and (ii) discarding proteins with undetermined or nuclear subcellular localization according to Uniprot annotation, and treating the remaining 259 non-nuclear proteins as negative examples (Figure 1— figure supplement 3A and Figure 1—source data 1B). In the former case, the reported cargo rate corresponds to a lower bound on the precision, and even in the latter case, the reported cargo rates are expected to underestimate precision, because almost certainly some of the proteins that we exclude as unclear are in fact true cargoes. To select cargoes with high sensitivity, we employed the cutoff of 15% that yields a high recall of 0.741 (Figure 1—figure supplement 3B and Figure 1—source data 1A and 1B; recall is not affected by the assumptions of negative examples). Among the 27 reported cargoes, 20 cargoes were ranked in the top 15% (247 proteins; p=5.39 Â 10À12 by Fisher’s exact test), and the others were dispersed in the lower ranks (Figures 1A and 2A; Figure 1—figure supplement 2C and E). We examined the direct binding of Trn-1 to a subset of proteins in the 2nd-Z-ranking using a bead halo assay (Patel and Rexach, 2008)(Supplementary file 2) in which the binding of GFP- fusion proteins to GST-Trn-1 on glutathione-Sepharose beads was observed by fluorescence microscopy. If RanGTP (a Q69L GTP-fixed mutant) (Bischoff and Ponstingl, 1995) inhibits the protein–Trn- 1 binding, the functionality of the binding is verified. For all the bead halo assays in this work, we principally selected well-characterized proteins that have not been reported as cargoes from (i) proteins ranked high (within the top 15% in the 2nd-Z-ranking or 4% in the 3rd-Z-ranking, see below), around presumptive cutoffs (within about top 15–25% in the 2nd-Z-ranking), or lower and (ii) highly ranked proteins that are suspected as indirect cargoes or false positives based on their well-known

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A B PY- PY- 2nd Z-score Rank 3rd Z-score NLS NLS Rate of PY-NLS proteins 5 4 3 2 1 0 -1 0 1 2 3 4 5 (% in 50 consecutive proteins) 1 Ranked by 2nd Z-score Ranked by 3rd Z-score Rank 10 40 30 20 10 0 0 10 20 30 40 50 1 20 100 30

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240 Basic PY-NLS: [Basic segment]X1–7[RKH]X2–6PY Basic segment: 5–17 aa Ratio of K, R or H >0.25 250 Both termini, K, R or H Reported Cargo: 27 Reported Cargo: 25 Quantified Protein: 1649 Quantified Protein: 1235 Hydrophobic PY-NLS : [FYKVRQP][GAS][VMPKSDE][VMSPQR]X [RKH]X PY Direct Binding: Blue, Positive ; Dark Gray, Negative; Gray, Not Determined 3–15 0–9

Figure 1. SILAC-Tp effectively sorts Trn-1 cargoes. (A) Z-scores in the Trn-1 2nd- and 3rd-Z-rankings. The second (left) and third (right) Z-scores are presented for the top 250 proteins in the Trn-1 2nd- and 3rd-Z-rankings, respectively. The total number of ranked (quantified) proteins and the number Figure 1 continued on next page

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Figure 1 continued of previously reported cargoes included in the ranking are indicated at the bottom. The magenta bars represent previously reported cargoes. The blue and dark gray bars represent the proteins that did and did not bind directly to Trn-1, respectively, in the bead halo assays (Supplementary file 2). Identical proteins marked by the colors are connected by lines. Proteins that carry PY-NLS motifs are indicated by green bars. (B) Distribution of PY-NLS motif-containing proteins in the rankings. The percentage of the proteins carrying PY-NLS motifs in 50 consecutively aligned proteins is presented along with the 2nd- and 3rd-Z-rankings (left and right, respectively). For example, the top 50 proteins in the 2nd-Z-ranking include 19 (38%) PY-NLS motif-containing proteins, and thus the value at position 1 is 38%. Two types of PY-NLS motifs, basic and hydrophobic, are defined as presented at the bottom. DOI: 10.7554/eLife.21184.002 The following source data and figure supplements are available for figure 1: Source data 1. Statistical analysis of reported cargoes in the Trn-1 2nd- and 3rd-Z-ranking. DOI: 10.7554/eLife.21184.003 Figure supplement 1. SILAC-Tp experimental system. DOI: 10.7554/eLife.21184.004 Figure supplement 2. Trn-1 cargoes are effectively sorted by the second or third Z-scores in three replicates of SILAC-Tp. DOI: 10.7554/eLife.21184.005 Figure supplement 3. Reported cargo rates and recall of the Trn-1 2nd- and 3rd-Z-ranking. DOI: 10.7554/eLife.21184.006

features, for example, S100A6 or EEF1A2 (see the legend of Supplementary file 2). The negative rate of the bead halo assays should be higher than the true overall false-positive rate of the SILAC- Tp, because proteins in (ii) are selected preferentially. Seventeen novel candidate cargoes in the top 266 (top 16%) bound to Trn-1, and RanGTP inhibited the binding (Figure 1A; Supplementary file 1, Trn-1 ‘Direct binding’; Supplementary file 2). Although the assays were not comprehensive, many of the highly ranked proteins are bona fide Trn-1 cargoes. The highly ranked proteins that did not bind to Trn-1 in the assays are still candidate indirect cargoes that may form complexes with other proteins that directly bind to Trn-1 (see the case of POLE3 for Imp-13 below). As an example of a protein with only a single but high Z-score (+NTR/Ctl value), DIMT1 bound to Trn-1 (DHRS4 with Imp-b is another example), but we did not consider such proteins. Because many reported Trn-1 cargoes carry PY-NLSs, we examined the distribution of PY-NLS motif-containing proteins in the 2nd-Z-ranking (Figure 1B). The percentages of PY-NLS motif-containing proteins within a window width of 50 positions were higher in the range of the top 200, indicating a higher rate of PY-NLS motif-containing proteins within the top 250 (top 15%). The reported Trn-1 cargoes were similarly distributed in the Trn-2 2nd-Z-ranking (Supplementary file 1, Trn-2 ‘Report or feature’). Because Trn-1 and -2 share nearly the same reported cargoes (Twyffels et al., 2014), this result demonstrates the reproducibility of the SILAC-Tp method. Based on these evalua- tions, we assumed that the proteins in the top 15% (247 proteins) of the 2nd-Z-ranking are candidate cargoes with high sensitivity (0.741) and termed them the 2nd-Z-15% cargoes. Next, we examined whether the cutoff employed for Trn-1 is applicable to Imp-13 and Trn-SR whose 2nd-Z-rankings include several reported cargoes. The Imp-13 2nd-Z-ranking (totaling 2060 proteins) includes eight reported cargoes (Supplementary file 1, Imp-13), and seven of these are ranked in the top 244 (top 12%; p=2.83 Â 10À7; Figure 2B; Figure 2—figure supplements 1A and 2A). In bead halo assays for a subset of the ranked proteins, 24 novel candidate cargoes in the top 326 (top 16%) bound directly to Imp-13, and RanGTP inhibited the binding (Figure 2—figure supplement 2A; Supplementary file 1, Imp-13; Supplementary file 2). One component of a reported cargo complex, that is, POLE3, did not bind to Imp-13, but its binding partner CHRAC1 (Walker et al., 2009) did. Thus, the binding partners of the direct cargoes are also ranked high. Many reported Trn-SR cargoes are SR-domain proteins (Chook and Su¨el, 2011), and they can be grouped into either SR-rich splicing factors (SFs) or other SR-domain proteins. The Trn-SR 2nd-Z- ranking (totaling 2021 proteins) contains three reported cargoes (Supplementary file 1, Trn-SR), and they are ranked in the top 55 (top 3%; p=1.91 Â 10À5; Figure 2C; Figure 2—figure supplement 2B). The 2nd-Z-ranking contains seven SR-rich SFs other than the reported SFs, and five of these are ranked in the top 90 (top 4%; p=7.61 Â 10À18). The 2nd-Z-ranking also contains another four proteins that are annotated with ‘RS-domain’ in UniProt, and three of these are ranked in the top 202 (top 10%; p=3.65 Â 10À3). Finally, in bead halo assays for a subset, 11 novel candidate cargoes in

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A Trn-1 B Imp-13 C Trn-SR Rank 2nd Z-score 3rd Z-score Rank 2nd Z-score 3rd Z-score Rank 2nd Z-score 3rd Z-score 1 ATF1 ATF1 1 NFYB NFYB 1 PMPCA SRRM2 2 KHDRBS1 KHDRBS1 2 KDM2A LRRC59 2 TNPO3 TRAP1 3 MRTO4 MRTO4 3 S100P GTF2A2 3 SRRM2 PHF6 4 HNRNPM HNRNPM 4 PRPSAP1 MAGOH 4 AIP SRSF6 5 TUBB3 BYSL 5 LRRC59 RBM8A 5 ERC1 SRSF8 6 HNRNPAB HNRNPA1 6 GTF2A2 CHRAC1 6 DCTN4 PLK1 7 HNRNPA1 HNRNPAB 7 UGGT1 POLE3 7 TRAP1 GOLIM4 8 HNRNPD ALYREF 8 MAGOH DDX27 8 SRSF2 AIP 9 BYSL HNRNPA3 9 RBM8A NFYA 9 THOC1 SRSF7 10 ALYREF HNRNPD 10 NOC2L NOC2L 10 PHF6 CNOT10 11 HNRNPA0 HNRNPA0 11 DHRS4 NQO2 11 PRPF38B SRSF2 12 QKI PABPN1 12 DDX27 SRSF8 12 KIF2C ZNF281 13 HNRNPA3 HEXIM1 13 CD3EAP TMEM43 13 ALDH18A1 DHX30 14 HEXIM1 EWSR1 14 UAP1 ARPC5 14 PAF1 ZC3H4 15 NUP205 HNRNPA2B1 15 GALE TXNDC17 15 ATP5E ACOT9 16 HNRNPA2B1 PQBP1 16 TPM2 SFPQ 16 SRSF6 DYNC1LI1 17 SLIRP ELAVL1 17 SHCBP1 CDC42BPB 17 ATAD2 SRGAP2 18 GOLGA2 ZNF787 18 PHF10 NAPRT1 18 SCAF8 SRSF1 19 EWSR1 HNRNPH2 19 CHRAC1 BOLA2 19 SRSF8 PLOD3 20 NFYA MACROD1 20 DIDO1 TIGAR 20 ACOT9 SUB1 21 PABPN1 FUS 21 OGDH RBM27 21 PLK1 PAF1 22 IVD PDLIM1 22 DDX23 COMT 22 GOLIM4 MAP7D1 23 TIMM13 UBQLN4 23 POLE3 RPL37 23 ITPR1 SAP18 24 PDLIM1 MAZ 24 CAPN1 SUMO2 24 KIF15 LSM14A 25 PBK HNRNPH1 25 ARHGEF7 RAVER1 25 SDHA NELFE 26 S100A6 PNN 26 AGPS UGGT1 26 SRSF7 CD2AP 27 EEF1A2 CLTA 27 ENAH MAP7 27 CCNB1 C9orf78 28 NOP56 TIGAR 28 UNC45A CAPN1 28 HADHA SLTM 29 CBX5 HNRNPF 29 PDHA1 SMARCE1 29 ZC3H4 CBX1 30 CARHSP1 PPP2R1B 30 PDCD11 ENOPH1 30 TARS2 WDR12 31 PQBP1 SAP18 31 AKAP13 ZYX 31 MLF2 CWF19L1 32 CPNE1 FAH 32 FDFT1 EEF2 32 XRN2 DBNL 33 AARS C5orf24 33 NFYA PRC1 33 CNOT10 DNAJB1 34 HNRNPH1 DNPEP 34 SRSF8 TIMM8A 34 PGRMC1 RBM12 35 SSBP1 EXOSC10 35 IPO4 WDR18 35 TTC9C TTK 36 DDT HNRPDL 36 MCCC2 CBS 36 DDX27 NAT10 37 COTL1 HIST1H4A 37 SLC9A3R1 PHF10 37 CBX1 DHX38 38 MT2A SMC3 38 UBE2O NAT10 38 ZNF281 UBR4 39 AKR1C1 FAHD1 39 MKI67 SUMO3 39 WBP11 DHX36 40 OLA1 DAZAP1 40 DYNC1LI1 GNPDA1 40 PDE3A DKC1 41 HNRNPH2 LONP1 41 PBK DBI 41 RPL7L1 ILKAP 42 EIF5A SMARCC1 42 TRMT10C BROX 42 POP4 CCNB1 43 LASP1 NOP56 43 CTTN EEFSEC 43 ADAR RPS6KA3 44 TMSB4X LRPAP1 44 METAP2 GOLGA4 44 ANXA3 TATDN1 45 ETFA HPD 45 CARHSP1 CLTC 45 SNX5 SRSF3 46 FUS MAGOH 46 ARPC1A IKBKAP 46 BLVRA PQBP1 47 TRIP13 BUD31 47 PELP1 G6PD 47 PYCR2 CDK2 48 NAP1L4 ARL6IP4 48 PDLIM5 GN=S100A13 48 AAAS DDX21 49 AKR1C3 MPST 49 TARS2 AHNAK 49 FUBP1 CSTF1 50 MCTS1 SUPT6H 50 YAP1 ZNF787 50 RBM25 DHX15 51 ELAVL1 PRPF38B 51 ATP6V1H GEMIN5 51 TK1 MPG 52 CMBL EXOSC5 52 SARS GRWD1 52 NADKD1 DNAJB4 53 ACTR2 BZW1 53 EIF4H SRSF2 53 PPIG CCDC101 54 GLRX3 NIT2 54 PDCD5 PPIE 54 CARHSP1 THOC1 55 ANXA11 NHP2 55 EDF1 SRSF3 55 SRSF1 PPP1CA 56 UQCRC2 CDC5L 56 EEF2 PELP1 56 RAVER1 RTFDC1 57 C5orf24 SHMT1 57 KTN1 TXLNG 57 EFHD2 FAM83D 58 PIR GRHPR 58 NNT UBE2I 58 SNX1 DDX5 59 ACTN1 EXOSC1 59 SH3BGRL APEH 59 EIF1AX PPIL1 60 EML4 POLR2I 60 NQO2 KRT8 60 MDC1 WDR3 61 CKS2 LACTB2 61 S100A4 LIN7C 61 DHX30 CPSF7 62 CKM WBP11 62 ENOPH1 UTP18 62 CPSF7 RAB8A 63 CNBP CHD4 63 ARPC5 PPP1R12A 63 SLC9A3R1 U2AF1 64 GFPT1 GSN 64 CLIC1 RPL11 64 COX4I1 HK2 65 PWP1 PGM2 65 CKM MARCKSL1 65 SAMHD1 LDB1 66 PFN1 TRNT1 66 AKR1C3 KRT17 66 SMN1 FTH1 67 STMN1 QDPR 67 AARS UFL1 67 ISOC1 ZRANB2 68 ACTN4 PPP6C 68 EIF5A HNRPDL 68 GTF2F1 SNX1 69 PRC1 ECM29 69 TUBB4B TXLNA 69 PES1 HMGB1 70 OTUB1 CTNNA1 70 AKR1C1 PABPC4 70 DHX38 CUL3 71 DSTN GDA 71 MYL12A SPAG9 71 SRGAP2 UBE2M 72 THOC1 PLIN3 72 PAWR ZC3HAV1 72 SRSF9 SEC13 73 BROX RBM8A 73 BOLA2 ARPC5L 73 SNX12 ACTR10 74 CLUH AIMP2 74 ARL6IP5 GSTM3 74 NAT10 PDHA1 75 CSRP1 COX17 75 TXLNA TXN 75 GBE1 REXO2 76 TPT1 NPM3 76 TIGAR IWS1 76 NUMA1 PPP5C 77 HINT1 RBMX 77 HN1 SMU1 77 NFKB1 EMG1 78 PDCD5 RANGAP1 78 CHMP4A OGDH 78 PLOD3 RAE1 79 NAP1L1 CDC73 79 REXO4 RALY 79 RTFDC1 CCDC6 80 MESDC2 TRMT1L 80 RAN RAB21 80 MRPL11 SIN3B 81 PSMD10 CDK11A 81 PLOD2 XRN2 81 DYNC1LI1 PRC1 82 SCLY SEC16A 82 CNBP GTF2F2 82 ANXA4 LSG1 83 CTPS1 CAND1 83 SRSF2 MYO1C 83 DDX21 ANXA4 84 COX17 PRDX5 84 EIF1AX HDAC3 84 MPST PPP1CC 85 ACTC1 TCEB2 85 IPO5 CWC15 85 DDX47 THOC3 86 ZNF787 TATDN1 86 NAPRT1 TPM3 86 CD2AP THYN1 87 PDLIM5 TBL1XR1 87 S100A6 NCBP2 87 SAP18 PFDN4 88 GBE1 EIF6 88 CSRP1 WRNIP1 88 ACO2 KIAA0101 89 SLC25A3 CIAPIN1 89 SUMF2 PSME4 89 GATAD2A PPIE 90 PCBP2 PRCC 90 EEF1A1 CLUH 90 SRSF3 HMGB3 91 TXN RARS 91 TAGLN2 RPL31 91 PPP1R12A CSNK1A1 92 CTTN MTA2 92 PLIN2 SNCG 92 CAMK2G SCFD1 93 PRPF6 NTPCR 93 PFN1 FLNB 93 PNN CKAP5 94 CDA MARS 94 ELAVL1 SRSF7 94 SMU1 FBL 95 HNRNPF AK2 95 BLVRA TUBG1 95 RAB13 SCRIB 96 MACROD1 HYOU1 96 STRAP PABPC1 96 NOP58 DBR1 97 MDN1 COA4 97 PXN DDX17 97 MED15 TPRKB 98 KPNA2 CNDP2 98 TMEM43 COA4 98 SUB1 FEN1 99 UBQLN4 SCLY 99 CDC42BPB PXN 99 DKC1 CSTF2 IBD 001 001 IBD PEPD 001 72MBR ELAVL1 100 CWF19L1 FKBP2

Figure 2. Trn-1, Imp-13, and Trn-SR cargo rankings. (A–C) The top 100 proteins in the Trn-1 (A), Imp-13 (B), and Trn-SR (C) 2nd- and 3rd-Z-rankings (left and right, respectively). Magenta, reported cargoes; blue, proteins bound directly to the NTR in the bead halo assays (Supplementary file 2); orange in (C), SR-rich SFs that have not been reported; and green in (C), other RS (SR)-domain proteins. Identical proteins marked by the colors are connected by lines. Figure 2 continued on next page

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Figure 2 continued DOI: 10.7554/eLife.21184.007 The following figure supplements are available for figure 2: Figure supplement 1. Imp-13 cargoes are effectively sorted by the second or third Z-scores in three replicates of SILAC-Tp. DOI: 10.7554/eLife.21184.008 Figure supplement 2. SILAC-Tp effectively sorts Imp-13 and Trn-SR cargoes. DOI: 10.7554/eLife.21184.009 Figure supplement 3. Imp-b cargo ranking and Z-scores in the 2nd- and 3rd-Z-rankings. DOI: 10.7554/eLife.21184.010

the top 237 (top 12%) bound directly to Trn-SR, and RanGTP inhibited the binding (Figure 2—figure supplement 2B; Supplementary file 1, Trn-SR; Supplementary file 2). Hence, the 2nd-Z-15% cargoes could also be defined for Imp-13 (309 proteins) and Trn-SR (302 proteins), and we applied this cutoff to the other NTRs that have few reported cargoes. The 2nd-Z-15% cargoes of the 12 NTRs are presented in Supplementary file 3. Some of the 2nd-Z-15% cargoes with low numbers of L/H counts showed deviation in Z-scores or L/H ratios in the three replicates of SILAC-Tp (Supplementary file 1), and an example of their quantitation qualities is presented in Supplementary file 4. Exceptionally, Imp-b uses Imp-a as an adaptor for cargo binding, and the cytosolic extract used for the transport system contained endogenous Imp-a. Four Imp-as were found in the Imp-b 2nd-Z- ranking (totaling 2027 proteins), and three of these are in the 2nd-Z-15% cargoes (p=1.19 Â 10À2; Supplementary file 1, Imp-b; Supplementary file 3). Thus, the Imp-b candidate cargoes must include both Imp-b-direct and Imp-a-dependent cargoes. Indeed, 31 proteins in the top 276 (top 14%) bound directly to Imp-a,-b, or both in the bead halo assays (Supplementary file 1, Imp-b; Fig- ure 2—figure supplement 3; Supplementary file 2). The border for the Imp-b candidate cargoes can be relaxed because Imp-b imports more cargoes than other NTRs with the help of Imp-a. Indeed, in the bead halo assays, many proteins in the top 35% of the 2nd-Z-ranking bound to Imp-a, although most of the proteins that bound directly to Imp-b were ranked in the top 259 (13%). Here, we employed the Imp-b 2nd-Z-15% cargoes (303 proteins) to enable equal comparisons with the cargoes of other NTRs.

Cargo selection with higher specificity Deviation of the LC-MS/MS quantification within the three replicates complicates cargo selection. However, the Z-scores of the highly ranked reported cargoes were reasonably high in all the three replicates possibly because many of the reported cargoes are abundant proteins that seldom pro- duce outliers in quantification (Figure 1—figure supplement 2; Figure 2—figure supplement 1). To select proteins that have high Z-scores in all the three replicates, we next ranked the proteins that had three +NTR/Ctl values by the third (lowest) Z-scores (3rd-Z-ranking). The reported cargo rates, recall, and p-values were calculated in 1% rank increments under two assumptions similarly to the case of 2nd-Z-ranking (Figure 1—figure supplement 3A and B and Figure 1—source data 1C and 1D). The reported cargo rate calculated under the assumption that proteins annotated with non-nuclear localization (178 proteins) are negative examples is as high as 0.85 at the cutoff of top 4% (Figure 1—figure supplement 3A and Figure 1—source data 1D). The Trn-1 3rd-Z-ranking (totaling 1235 proteins) included 25 reported cargoes, and 17 of these were ranked in the top 37 (top 3%; p=1.67 Â 10À22; Figures 1A and 2A; Figure 1—figure supplement 2D and F; Supplementary file 1). Seven proteins in the top 47 (top 4%) were novel Trn-1-direct cargoes that were verified in the bead halo assays (Figures 1A and 2A; Supplementary files 1 and 2). The percentage of PY-NLS motif-containing proteins within a window width of 50 positions was highest at the first position (Figure 1B), indicating that PY-NLS motif-containing proteins are concentrated in the top 50 (top 4%). Thus, most of the proteins that ranked in the top 4% (49 proteins) of the 3rd-Z- ranking are highly reliable cargoes, and we termed these proteins the 3rd-Z-4% cargoes. In a com- parison between the Trn-1 2nd-Z-15% and 3rd-Z-4% cargoes, most of the 3rd-Z-4% cargoes were also 2nd-Z-15% cargoes (Figure 1A). Some reported or newly identified cargoes in the 2nd-Z-15% cargoes were ranked lower in the 3rd-Z-ranking due to the deviations in the third Z-scores.

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 7 of 31 Tools and resources Biochemistry Cell Biology In the Imp-13 3rd-Z-ranking (totaling 1671 proteins), seven proteins were reported cargoes, and six of these were ranked in the top 58 (top 3%; p=9.20 Â 10À9; Figure 2B; Figure 2—figure supplements 1B and 2A; Supplementary file 1). Additionally, the 3rd-Z-4% cargoes (66 proteins) included eight novel cargoes that directly bound to Imp-13 (Figure 2B; Figure 2—figure supplement 2A; Supplementary files 1 and 2). In the Trn-SR 3rd-Z-ranking (totaling 1591 proteins), both of the two reported cargoes were ranked in the top 18 (top 1%; p=1.21 Â 10À4), four of the five other SR-rich SFs were in the top 45 (top 3%; p=2.74 Â 10À6), one of the three SR-domain proteins (other than the SR-rich SFs) was ranked 63rd (top 4%; p=0.11), and six novel cargoes within the top 4% (63 proteins) bound directly to Trn-SR (Figure 2C; Figure 2—figure supplement 2B; Supplementary files 1 and 2). In cases of both Imp-13 and Trn-SR, the proteins were replaced between the 2nd- and 3rd- Z-rankings in a manner similar to the case for Trn-1. We concluded that the 3rd-Z-4% criteria is highly specific including few false positives, albeit at the cost of losing many genuine cargoes. Hence, we employed the 3rd-Z-4% cargoes mainly for the characterization of the identified cargoes, whereas the 2nd-Z-ranking was used for the evaluation of the import efficiencies of the expected cargoes. The 3rd-Z-4% cargoes of the 12 NTRs are presented in Figure 3.

Redundancy in the import pathways A total of 468 proteins were identified as 3rd-Z-4% cargoes of the 12 NTRs, and 332 of these are unique to one NTR, which clearly reflects the division of roles among the NTRs (Supplementary file 5B). Another 136 proteins were shared by two to seven NTRs, and the mean number of shared cargoes between two NTRs was 4.8. In the maximum-likelihood phylogenetic tree of the 12 NTRs (Figure 4A), Trn-1 and -2 (84% sequence identity) are paired most closely, and Imp-7 and -8 (65% identity) are the second-most closely paired. These paired NTRs share 28 and 19 cargoes, respectively, and they are paired similarly in a hierarchical clustering based on the cargo profiles (Figure 4B and C). The other NTRs that were paired weakly in the phylogenetic tree, namely, Imp- 13 and Trn-SR (23% identity), Imp-4 and -5 (22% identity), and Imp-9 and -11 (19% identity), did not form the same pairs when clustering by their cargoes. Thus, the NTR–cargo interactions are con- served only within the highly homologous NTRs. The 2nd-Z-15% cargoes included as many as 1416 proteins in total, 827 of which are shared by two to 12 NTRs, and 589 are unique to one NTR (Supplementary file 5A and 5D). Imp-7 and -8 share the largest number (162) among the 2nd-Z- 15% cargoes, but Trn-1 and -2 share no more than the other pairs. Of the 247 Trn-1 and 246 Trn-2 2nd-Z-15% cargoes, 69 are shared, and 36 of these are ranked within the top 50 in either ranking. Thus, Trn-1 and -2 still share many highly ranked cargoes but few lower ranked cargoes within the top 15%. The import efficiency of a cargo may differ between Trn-1 and -2, and only one of Trn-1 or -2 may import inefficient cargoes that are ranked lower.

Division of roles among the NTRs Because NTR-dependent transport is regulated, a cargo cohort of an NTR must be imported simultaneously and act cooperatively. To explore the roles of the NTR cargoes, the 3rd-Z-4% and 2nd-Z- 15% cargoes of each NTR were analyzed for enrichment of Gene Ontology (GO) terms (Gene Ontology Consortium, 2015) using g:Profiler (Reimand et al., 2016). For all the combinations of a GO term and an NTR, the number of cargoes annotated with the term and the significance (p-values according to g:SCS) of the term enrichment are listed (Supplementary files 6B, 6C, 7, and 8). Depending on the hierarchy of the GO terms, the terms are significantly annotated (p<0.05) to the cargo cohorts of none to 12 of the NTRs. Broader terms with smaller term depths are linked to more NTRs, whereas more defined terms with larger term depths are linked to fewer NTRs. Indeed, all 12 of the NTRs are linked to many broad terms, although the cargo numbers and the significances vary widely. Because similar terms were listed redundantly, we selected representative GO terms from those enriched significantly (p<0.05) for the 3rd-Z-4% cargoes of the 12 NTRs and tabulated the correspondences between the cargoes and the annotated terms (Supplementary file 9). To compare the GO terms that are specifically linked to each NTR, we listed the terms that are enriched significantly for the cargoes of four or fewer NTRs (Figures 5 and 6). Here, again we extracted the representative terms to decrease the size of the list. The selected terms for the 3rd-Z-4% cargoes plainly exhibit the roles of the cargo cohorts. For example, significant numbers of Imp-4, -7 and Exp- 4 cargoes are annotated with DNA recombination or DNA conformation (geometric) change

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Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Z-score Z-score Z-score Z-score Z-score Z-score Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 1 1 KPNB1 1 1 ATF1 1 1 ATF1 1 3 SRRM2 1 2 IPO4 1 1 IPO5 2 2 RNMT 2 2 KHDRBS1 2 10 ETFA 2 7 TRAP1 2 8 PFKL 2 11 NFYA 3 3 FAM103A1 3 3 MRTO4 3 4 KHDRBS1 3 10 PHF6 3 10 BMS1 3 17 DDX56 4 4 KIFC1 4 4 HNRNPM 4 5 HNRNPM 4 16 SRSF6 4 3 TFRC 4 42 HK2 5 5 TSR1 5 9 BYSL 5 9 MRTO4 5 19 SRSF8 5 18 MFAP1 5 26 TP53I3 6 20 LRRC59 6 7 HNRNPA1 6 7 HNRNPA1 6 21 PLK1 6 25 ALPP 6 27 BOP1 7 19 NCBP2 7 6 HNRNPAB 7 14 HNRNPA3 7 22 GOLIM4 7 32 FKBP10 7 6 THOC1 8 25 NCBP1 8 10 ALYREF 8 13 HNRNPA0 8 4 AIP 8 55 NELFE 8 132 SPIN1 9 27 ALKBH5 9 13 HNRNPA3 9 16 ALYREF 9 26 SRSF7 9 17 RBMX 9 146 FBL 10 21 NUP214 10 8 HNRNPD 10 12 HNRNPAB 10 33 CNOT10 10 31 SFPQ 10 117 C5orf24 11 37 SRRT 11 11 HNRNPA0 11 22 PABPN1 11 8 SRSF2 11 90 CALU 11 156 PDS5B 12 30 NUP88 12 21 PABPN1 12 8 HNRNPD 12 38 ZNF281 12 19 SLTM 12 189 RBM39 13 35 GTF2F1 13 14 HEXIM1 13 23 EWSR1 13 61 DHX30 13 88 ESYT1 13 52 LSM14A 14 22 HELLS 14 19 EWSR1 14 34 DECR1 14 29 ZC3H4 14 115 HNRNPUL2 14 165 ENOPH1 15 53 API5 15 16 HNRNPA2B1 15 19 HNRNPA2B1 15 20 ACOT9 15 42 PLK1 15 44 DIDO1 16 58 CHD8 16 31 PQBP1 16 69 UBTF 16 81 DYNC1LI1 16 89 CMAS 16 238 CDC42BPB 17 70 POLD3 17 51 ELAVL1 17 42 HNRNPH2 17 71 SRGAP2 17 12 PICALM 17 195 XRN2 18 46 RCC1 18 86 ZNF787 18 43 FUS 18 55 SRSF1 18 119 RCC1 18 175 U2AF1 19 45 ZNF787 19 41 HNRNPH2 19 29 HEXIM1 19 78 PLOD3 19 101 PRCC 19 131 MAP7D1 20 23 MYOF 20 96 MACROD1 20 31 PQBP1 20 98 SUB1 20 13 BCLAF1 20 150 SNX5 21 33 MTA1 21 46 FUS 21 37 ELAVL1 21 14 PAF1 21 111 PPIL3 21 278 DEK 22 82 SHMT2 22 24 PDLIM1 22 30 NOP56 22 130 MAP7D1 22 67 HSPA9 22 142 RAVER1 23 84 ALYREF 23 99 UBQLN4 23 40 HNRNPH1 23 87 SAP18 23 129 DHX9 23 94 MFAP1 24 61 GTF2F2 24 119 MAZ 24 91 CTR9 24 118 LSM14A 24 130 HMGB1 24 76 TUFM 25 94 CTCF 25 34 HNRNPH1 25 123 ATP2A2 25 123 NELFE 25 131 FTH1 25 22 NADKD1 26 73 NOP9 26 162 PNN 26 28 PRPF6 26 86 CD2AP 26 165 RAB7A 26 145 CPSF7 27 67 ZNF24 27 122 CLTA 27 17 UTP18 27 141 C9orf78 27 71 CHCHD2P9 27 51 PTPN1 28 95 PMS2 28 195 TIGAR 28 52 HNRNPF 28 101 SLTM 28 147 DNAJC8 28 308 GBE1 29 60 WDHD1 29 95 HNRNPF 29 45 APRT 29 37 CBX1 29 211 RPL36AL 29 119 GNB2 30 109 MAZ 30 182 PPP2R1B 30 206 ARL6IP4 30 223 WDR12 30 92 SHMT2 30 368 CD44 31 66 LDB1 31 206 SAP18 31 185 USP39 31 100 CWF19L1 31 163 ISOC1 31 184 PHF6 32 127 MNAT1 32 279 FAH 32 50 C5orf24 32 204 DBNL 32 180 ZMYM3 32 153 MBD2 33 144 ZNF146 33 57 C5orf24 33 65 MAZ 33 198 DNAJB1 33 83 H1FX 33 188 DDX46 34 100 TPX2 34 319 DNPEP 34 300 SAP18 34 159 RBM12 34 191 GOT2 34 226 ZNF146 35 26 OGDH 35 192 EXOSC10 35 105 BUD31 35 180 TTK 35 262 AQR 35 12 SNX12 36 149 CCDC86 36 134 HNRPDL 36 228 DAZAP1 36 74 NAT10 36 214 KIF1BP 36 420 GAR1 37 128 POLR3C 37 187 HIST1H4A 37 416 RPL32 37 70 DHX38 37 229 GTF2F2 37 418 LUC7L 38 119 THYN1 38 147 SMC3 38 450 RPL22 38 108 UBR4 38 246 PCID2 38 182 SNRNP27 39 97 SMARCE1 39 331 FAHD1 39 383 ATL3 39 295 DHX36 39 269 SCFD1 39 218 U2AF2 40 80 POLD2 40 233 DAZAP1 40 239 RAB9A 40 99 DKC1 40 79 PLIN2 40 326 MAZ 41 48 CYR61 41 276 LONP1 41 217 RAB7A 41 219 ILKAP 41 179 CDKN2A 41 114 PLOD3 42 155 SPIN1 42 378 SMARCC1 42 272 LACTB2 42 27 CCNB1 42 198 CWF19L1 42 380 SRSF7 43 121 CCDC58 43 28 NOP56 43 287 CTNND1 43 177 RPS6KA3 43 173 HMGB3 43 367 HSPA9 44 180 HMGA1 44 116 LRPAP1 44 144 ZYX 44 235 TATDN1 44 94 MATR3 44 370 ISOC1 45 130 LMNA 45 499 HPD 45 425 PRRC2A 45 90 SRSF3 45 37 EIF1AX 45 379 SRSF3 46 86 SLTM 46 388 MAGOH 46 85 HNRNPH3 46 150 PQBP1 46 207 HMGB2 46 449 RAB31 47 173 U2SURP 47 124 BUD31 47 491 RPL11 47 353 CDK2 47 272 POLR2L 47 510 SDHA 48 185 TCERG1 48 321 ARL6IP4 48 417 RPL23A 48 83 DDX21 48 123 RFC5 48 369 SRSF8 49 189 PES1 49 376 MPST 49 141 PNN 49 384 CSTF1 49 24 AKAP8 49 344 PLRG1 50 145 HNRPLL 50 255 ACSL4 50 406 DHX15 50 110 GPKOW 50 451 NUCKS1 51 81 KPNA2 51 161 MPG 51 514 DDX17 52 188 HMGN3 52 336 DNAJB4 52 479 LUC7L2 53 57 RBM27 53 416 CCDC101 53 137 HNRNPA0 54 29 KHSRP 54 9 THOC1 54 155 ACTR10 55 191 SUMF2 55 318 PPP1CA 55 306 RPL22 56 222 HNRPDL 56 79 RTFDC1 56 357 ARL6IP4 57 225 TAF15 57 275 FAM83D 57 271 UBE4B 58 263 DKC1 58 116 DDX5 58 275 PLIN2 59 141 HNRNPUL2 59 466 PPIL1 59 477 HNRNPL 60 219 C9orf78 60 266 WDR3 60 527 PRC1 61 79 MRGBP 61 62 CPSF7 61 500 SIN3B 62 123 HNRNPU 62 320 RAB8A 62 375 RBM26 63 75 OAT 63 202 U2AF1 63 466 ADAR 64 168 ATP5A1 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Z-score Z-score Z-score Z-score Z-score Z-score Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name Rank Gene Name 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 3rd 2nd 1 2 TRAP1 1 4 ZNF787 1 3 H2AFZ 1 9 RALY 1 1 NFYB 1 3 DDX39B 2 9 ZNF787 2 6 TFRC 2 1 ACAT1 2 12 TFRC 2 5 LRRC59 2 4 DDX39A 3 25 ERP44 3 17 HSPD1 3 13 CYR61 3 34 AKR1C1 3 6 GTF2A2 3 9 JUP 4 23 HMGB2 4 12 NOP56 4 16 RALY 4 24 EIF4A3 4 8 MAGOH 4 23 VIM 5 37 HIST1H4A 5 3 RALY 5 4 FUBP1 5 53 RBM25 5 9 RBM8A 5 25 XRN2 6 47 HNRNPUL1 6 34 MAZ 6 5 RBMX 6 11 ACO2 6 19 CHRAC1 6 8 TMPO 7 29 ZNF207 7 40 RPL11 7 44 GOT2 7 36 ALDH18A1 7 23 POLE3 7 24 HSPD1 8 51 ACADVL 8 42 RPL22 8 40 IVD 8 91 ACP1 8 12 DDX27 8 35 ALDH1B1 9 63 BUB3 9 28 GOT2 9 45 TFRC 9 43 ESYT2 9 33 NFYA 9 31 CS 10 16 VIM 10 7 HIST1H4A 10 18 CS 10 47 RBM39 10 10 NOC2L 10 67 TFRC 11 40 NUP155 11 36 SLC25A1 11 7 PRPF6 11 120 GLOD4 11 60 NQO2 11 7 SLTM 12 80 DIDO1 12 60 ZNF24 12 31 HSPA9 12 41 LRRFIP1 12 34 SRSF8 12 34 TOMM70A 13 53 HMGB3 13 25 NNT 13 9 HNRNPUL1 13 124 MACROD1 13 98 TMEM43 13 96 PSIP1 14 44 FKBP10 14 88 ARMC1 14 12 SUPT16H 14 140 CKAP5 14 63 ARPC5 14 13 NOP56 15 65 ZNF146 15 89 RAB14 15 84 BMS1 15 25 IL18 15 174 TXNDC17 15 75 ALYREF 16 72 GOT2 16 19 DDX21 16 75 ZNF787 16 173 RDX 16 143 SFPQ 16 82 HNRNPA0 17 83 ZNF24 17 67 RAB1A 17 55 KMT2A 17 185 MSN 17 99 CDC42BPB 17 120 GTF2I 18 70 SDHA 18 77 RBM3 18 78 MAZ 18 49 AFG3L2 18 86 NAPRT1 18 89 FAM32A 19 39 SHMT2 19 21 NFYA 19 47 SHMT2 19 127 LGALS1 19 73 BOLA2 19 98 ZNF638 20 102 H1FX 20 99 RAB2A 20 25 DHX30 20 116 TMA7 20 76 TIGAR 20 103 CPSF6 21 105 SRSF1 21 43 SNX27 21 151 SRRM2 21 177 U2AF1 21 100 RBM27 21 78 RBMX 22 68 SRSF2 22 61 RAB8A 22 146 SART1 22 143 NEDD8 22 111 COMT 22 105 SUMF2 23 115 LUC7L3 23 63 FKBP10 23 157 H1F0 23 62 HIST1H4A 23 130 RPL37 23 122 RRBP1 24 89 OXCT1 24 103 LGALS1 24 62 PDHB 24 172 DTYMK 24 118 SUMO2 24 115 FBL 25 86 SRSF7 25 78 RAB21 25 191 PRPF3 25 187 APRT 25 226 RAVER1 25 135 EXOSC1 26 173 PRPF6 26 10 SHMT2 26 130 WDR74 26 167 LANCL2 26 7 UGGT1 26 42 SFPQ 27 67 FXR1 27 106 GOLPH3 27 211 RPL27A 27 108 MRTO4 27 224 MAP7 27 134 THBS1 28 148 RSRC2 28 127 DDX3X 28 30 JUP 28 216 VPS45 28 24 CAPN1 28 136 RBM26 29 132 MAZ 29 101 RAB1B 29 131 ACAA2 29 221 NCOR2 29 234 SMARCE1 29 156 NONO 30 217 SF3B5 30 93 RAB10 30 23 DDX17 30 110 TBL1XR1 30 62 ENOPH1 30 155 LUC7L2 31 50 TUFM 31 125 HK1 31 81 RPS7 31 186 ARHGDIA 31 250 ZYX 31 62 CPSF7 32 166 CDC5L 32 146 THBS1 32 194 PPP1R10 32 255 RPL35A 32 56 EEF2 32 193 CDC73 33 146 ACIN1 33 29 CCAR1 33 229 SF3B5 33 162 CLINT1 33 132 PRC1 33 70 ARL6IP4 34 81 SRSF11 34 134 LUC7L3 34 92 ALYREF 34 121 ACYP1 34 279 TIMM8A 34 66 U2AF1 35 207 SNRPA1 35 148 U2AF1 35 166 WBSCR22 35 98 RAD23A 35 292 WDR18 35 93 RBM39 36 208 ARL6IP4 36 20 VIM 36 43 MYOF 36 215 TXNL1 36 172 CBS 36 214 ERH 37 183 SNRPB2 37 165 NUDT21 37 214 DDX24 37 101 GDI2 37 18 PHF10 37 61 WDR33 38 219 CDK12 38 149 ACIN1 38 149 YY1 38 189 KTN1 38 195 NAT10 38 99 GAR1 39 26 HSPD1 39 120 SRSF7 39 165 RPL23A 39 26 MYL12A 39 309 SUMO3 39 189 CD55 40 206 SF3A1 40 177 HNRNPH3 40 193 RPL35A 40 286 U2AF2 40 278 GNPDA1 40 153 MAP7 41 97 SLC27A2 41 191 ELAVL1 41 208 NUP98 41 3 JUP 41 157 DBI 41 170 SUMO1 42 155 HMGB1 42 39 U2SURP 42 284 LMNB1 42 52 LPP 42 228 BROX 42 39 HIST1H4A 43 204 ARGLU1 43 166 MYO1B 43 250 PPIB 43 70 CCBL2 43 377 EEFSEC 43 177 HNRNPA1 44 253 RAB31 44 197 ACSL4 44 170 CELF1 44 73 DDX46 44 148 GOLGA4 44 171 PTBP1 45 295 RPN1 45 128 U2AF2 45 205 RPL15 45 318 SERPINB6 45 379 CLTC 45 233 RAP1B 46 151 GNB1 46 62 RAB13 46 274 SSRP1 46 200 TBCB 46 324 IKBKAP 46 147 RAB35 47 140 IFI16 47 118 SRSF3 47 54 BCLAF1 47 223 RPL34 47 384 G6PD 47 146 SCAF11 48 117 SNRPA 48 217 SF3B4 48 156 SNRPA1 48 328 BTF3 48 189 S100A13 48 53 ARGLU1 49 165 HNRNPA3 49 196 MYO1C 49 73 CD55 49 304 RPL18A 49 412 AHNAK 49 92 SRSF3 50 177 SF3A2 50 193 G3BP1 50 262 RPL7A 50 85 RTF1 50 316 ZNF787 51 282 SF3B2 51 172 DDX6 51 88 BBX 51 153 GALK1 51 406 GEMIN5 52 22 NNT 52 50 HNRNPUL1 52 128 RPL36 52 349 NPEPPS 52 233 GRWD1 53 260 SNRPD3 53 147 SF3B1 53 326 RPL27 53 83 SRSF2 54 240 RAP1B 54 136 RPL30 54 453 PPIE 55 311 SF3B4 55 207 SF3B5 55 357 SRSF3 56 286 THBS1 56 69 RAB31 56 47 PELP1 57 222 SF3A3 57 153 EIF3L 57 215 TXLNG 58 228 SNRPB 58 91 FIS1 58 244 UBE2I 59 81 SRSF11 59 478 APEH 60 221 SUB1 60 425 KRT8 61 475 LIN7C 62 229 UTP18 63 231 PPP1R12A 64 401 RPL11 65 162 MARCKSL1 66 397 KRT17

Figure 3. 3rd-Z-4% cargoes of the 12 NTRs. The 3rd-Z-4% cargoes of each NTR are listed by the gene names in the 3rd-Z-rank orders. The ranks by the second Z-scores are also shown. The 3rd-Z-4% and 2nd-Z-15% cargoes are indicated by cyan in the rank columns. Colors in the gene name columns: magenta, reported cargoes; blue, cargoes bound directly to the NTR in the bead halo assays (Supplementary file 2); light blue, cargoes bond directly to Imp-a but not to Imp-b; gray, proteins that did not bind to the NTRs; and yellow, Imp-a. For the 2nd-Z-15% cargoes, see Supplementary file 3. DOI: 10.7554/eLife.21184.011

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A B 65.5 12

11 100 Imp-13 54.9 36.5 Trn-SR 62 10 0.1 Imp-13 79.8 Imp-11 Trn-SR

82.2 9 Exp-4 Exp-4 Imp-5 Imp-4 Imp-9 Imp-7 Imp-8 100 8 Trn-1 Trn-2 100 7 Imp-11 Imp-9 Imp-8 Imp-7 Imp-4 Imp- 6 Trn-2 Trn-1 Imp-5 β

C Total in Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Unique 3rd-Z-4% Imp- 4 2 3 5 3 5 5 6 0 4 3 42 63 Trn-1 28 2 0 4 5 5 3 3 3 6 15 49 Trn-2 2 1 5 4 7 4 2 3 5 12 50 Trn-SR 4 10 4 6 2 1 4 4 36 63 Imp-4 4 7 4 7 1 1 4 25 49 Imp-5 8 8 3 4 7 11 23 62 Imp-7 19 8 1 2 7 22 58 Imp-8 8 6 4 8 24 60 Imp-9 4 1 6 28 53 Imp-11 0 5 39 52 Imp-13 3 46 66 Exp-4 20 49

Figure 4. Phylogenetic tree and cargo profile hierarchical clustering of the Imp-b family import receptors. (A) Phylogenetic tree of the 12 Imp-b family import receptors with the bootstrap values. Scale bar indicates substitutions per site. (B) A hierarchical clustering dendrogram of the same NTRs (except Imp-b) based on the similarities of their 3rd-Z-4% cargo profiles. Imp-b was excluded because Imp-a connects to Imp-b and many of the identified cargoes. The scale indicates the intercluster distance. (C) The numbers of 3rd-Z-4% cargoes shared by two NTRs. For the 2nd-Z-15% cargoes, see Supplementary file 5A. DOI: 10.7554/eLife.21184.012

(Figure 5; Supplementary file 9), which are terms for biological processes (BPs). These cargoes are also annotated with chromatin, which is a term for cellular component (CC; Figure 6A; Supplementary file 9), and DNA binding, which is for molecular function (MF; Figure 6B; Supplementary file 9), all related to DNA recombination and DNA conformation change. For another example, the Trn-SR cargoes are significantly annotated with a range of terms for BPs that are related to cell division or nuclear division and terms for CCs that include condensed chromosome, kinetochore, spindle, and centrosome. Similarly, most of the examined NTRs are linked to terms for BPs via the 3rd-Z-4% cargoes (Figure 5) as follows: Imp-b, -4, -7, and Trn-SR are linked to chromatin or chromosome organization; Imp-b, -4, and -13 are linked to DNA repair; Imp-b is linked to mRNA capping; Trn-SR and Exp-4 are linked to mRNA polyadenylation; Trn-1 and -2 are linked to mRNA stabilization; Trn-2, -SR, Imp-5, -9, and Exp-4 are linked to ribosome biogenesis or rRNA processing; Trn-SR is linked to protein folding, modification, ubiquitination, and catabolic process; and Imp-4 and -7 are linked to apoptosis. The NTRs are also consistently linked to the terms for CCs and MFs (Figure 6) as follows: Trn-SR, Imp-5, -9, and Exp-4 are linked to Cajal body; Imp-b is linked to cap-binding complex; and Trn-SR is linked to pre-mRNA binding, snoRNA binding, and RNA helicase activity.

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Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Total Term ID Term Name p # p # p # p # p # p # p # p # p # p # p # p # No. GO:0006259 DNA Metabolic Process 8E-06 11 0.199 6 1 2 0.146 7 0.002 8 1 4 0.632 6 1 2 0.34 6 1 2 2E-04 10 8E-06 10 978 GO:0006310 DNA Recombination 0.503 4 1 1 1 2 0.008 5 1 1 0.019 5 1 2 1 1 1 1 1 3 0.008 5 298 GO:0006323 DNA Packaging 1 3 1 1 1 2 0.039 4 0.003 5 1 2 1 1 1 1 1 1 1 1 200 GO:0071103 DNA Conformation Change 0.312 4 1 1 1 2 1E-04 6 4E-04 6 0.244 4 1 1 1 1 1 1 1 1 263 GO:0032392 DNA Geometric Change 1 2 4E-04 4 0.061 3 1 2 63 GO:0010216 Maintenance of DNA Methylation 0.031 2 6 GO:0000737 DNA Catabolic Process, Endonucleolytic 0.002 3 0.436 2 1 1 23 GO:0006281 DNA Repair 0.002 7 1 3 1 2 0.007 6 1 1 1 4 1 1 1 3 1 2 0.043 6 0.111 5 519 GO:0006289 Nucleotide-Excision Repair 0.475 3 0.005 4 1 1 1 1 0.017 4 1 1 116 GO:0006284 Base-Excision Repair 0.043 3 1 1 1 2 1 1 52 GO:0006325 Chromatin Organization 6E-07 11 1 3 1 3 0.313 6 0.062 6 1 5 1 4 1 1 0.943 5 1 4 0.374 6 1 4 768 GO:0031497 Chromatin Assembly 1 3 1 1 1 1 0.017 4 0.035 4 1 1 1 1 1 1 1 1 1 1 162 GO:0016568 Chromatin Modification 2E-05 9 1 2 1 1 0.967 5 1 3 0.967 5 1 1 1 1 1 2 1 3 1 5 1 4 614 GO:0006338 Chromatin Remodeling 0.037 4 1 2 1 1 1 1 1 1 1 1 1 1 1 2 1 1 152 GO:0070828 Heterochromatin Organization 7E-04 3 1 1 14 GO:0051276 Chromosome Organization 4E-11 16 1 5 1 4 3E-06 12 2E-06 11 0.348 7 0.021 8 1 4 0.723 6 1 4 0.43 7 0.497 6 1126 GO:0032200 Telomere Organization 0.031 4 1 2 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 0.414 3 145 GO:0006368 Transcription Elongation from RNA Polymerase 1E-04 5 1 1 1 2 0.118 3 1 2 1 1 1 2 1 1 95 II Promoter GO:0016458 Gene Silencing 1E-05 7 1 2 1 2 1 2 1 1 1 2 1 2 1 2 1 3 1 1 1 1 244 GO:0034660 ncRNA Metabolic Process 3E-06 9 0.986 4 1 4 0.312 5 1 2 0.023 6 1 3 1 3 0.107 5 1 2 0.358 5 1 4 480 GO:0034470 ncRNA Processing 0.046 5 0.241 4 0.307 4 0.054 5 1 1 0.054 5 1 1 1 3 0.018 5 1 2 0.954 4 0.272 4 331 GO:0006370 7-Methylguanosine mRNA Capping 6E-12 7 0.616 2 33 GO:0000387 Spliceosomal snRNP Assembly 1 2 1 1 0.017 3 0.012 3 1 1 41 GO:0000389 mRNA 3'-Splice Site Recognition 3E-05 3 6 GO:0006378 mRNA Polyadenylation 1 1 0.018 3 1 1 1 1 0.007 3 37 GO:0098789 pre-mRNA Cleavage Required for 0.031 2 6 Polyadenylation GO:0000184 Nuclear-Transcribed mRNA Catabolic Process, 1 2 1 2 0.006 4 1 1 0.454 3 3E-10 8 0.008 4 0.021 4 123 Nonsense-Mediated Decay GO:0048255 mRNA Stabilization 1 1 0.005 3 0.006 3 1 1 1 1 1 1 1 1 34 GO:0010501 RNA Secondary Structure Unwinding 0.032 3 0.032 3 0.023 3 1 2 1 2 1 1 1 2 45 GO:0042254 Ribosome Biogenesis 0.203 4 0.064 4 0.003 5 4E-04 6 1 2 0.236 4 0.158 4 5E-08 8 0.1 4 0.26 4 0.073 4 235 GO:0006364 rRNA Processing 1 2 0.456 3 0.557 3 0.001 5 1 1 0.047 4 0.894 3 0.019 4 1 2 0.052 4 0.014 4 155 GO:0042273 Ribosomal Large Subunit Biogenesis 1 1 1 2 1 2 1 1 1 1 0.014 3 1 1 1 1 43 GO:0006412 Translation 1 3 0.333 5 1E-04 8 1 5 1 2 1 4 0.895 5 2E-06 10 1E-05 9 1 4 0.001 8 1 3 677 GO:0006413 Translational Initiation 1 2 1 1 0.006 5 1 1 1 1 1 1 0.014 5 2E-07 8 1 3 1 2 273 GO:0006414 Translational Elongation 0.062 4 1 2 1 1 1 3 3E-08 8 0.076 4 0.198 4 219 GO:0006415 Translational Termination 0.028 4 1 1 1 3 6E-09 8 0.973 3 1 3 179 GO:0006457 Protein Folding 1 1 0.012 5 1 3 1 1 0.162 4 1 2 1 2 1 2 1 2 1 1 240 GO:0061077 Chaperone-Mediated Protein Folding 1 2 1 1 0.032 3 1 2 1 1 1 1 51 GO:0010608 Posttranscriptional Regulation of Gene 8E-04 7 0.003 6 0.068 5 0.017 6 1 3 1 3 1 3 0.01 6 1 1 1 1 0.277 5 0.902 4 454 Expression GO:0043412 Macromolecule Modification 0.024 15 1 7 1 5 2E-04 18 0.617 11 1 10 0.807 12 1 7 1 6 1 9 0.832 13 0.617 11 4139 GO:0036211 Protein Modification Process 1 11 1 6 1 4 0.004 16 0.42 11 1 9 0.538 12 1 6 1 6 1 9 0.542 13 1 9 3959 GO:0016567 Protein Ubiquitination 1 1 1 1 0.036 7 1 3 1 1 1 2 1 1 1 1 1 1 784 GO:0018205 Peptidyl-Lysine Modification 0.007 6 1 1 1 1 1 2 1 2 1 1 1 2 1 1 0.149 5 1 1 398 GO:0031145 Anaphase-Promoting Complex-Dependent 4E-04 5 1 2 1 2 120 Proteasomal Ubiquitin-Dependent Protein Catabolic Process GO:0030163 Protein Catabolic Process 1 1 1 3 1 1 0.005 8 1 4 1 2 1 2 1 5 1 3 1 1 827 GO:0007049 Cell Cycle 5E-05 13 1 4 1 3 0.004 11 0.15 8 1 6 1 7 1 7 1 2 1 4 1 6 0.15 8 1796 GO:0000278 Mitotic Cell Cycle 2E-04 10 1 3 1 1 0.025 8 0.033 7 1 3 1 5 1 5 1 1 1 3 1 4 1 2 1038 GO:0051301 Cell Division 1 4 1 1 1 1 0.001 8 1 4 1 2 1 2 1 1 1 1 1 2 1 1 672 GO:0000075 Cell Cycle Checkpoint 5E-04 6 1 3 1 1 1 3 1 1 249 GO:0071173 Spindle Assembly Checkpoint 4E-04 4 1 2 1 2 46 GO:0007091 Metaphase/Anaphase Transition of Mitotic Cell Cycle 1E-03 4 1 2 1 2 59 GO:0007059 Chromosome Segregation 1 3 1 1 0.024 5 0.141 4 1 1 1 2 1 1 1 2 1 1 279 GO:0000280 Nuclear Division 0.042 6 1 1 0.004 7 1 4 1 1 1 2 1 1 1 3 550 GO:0007077 Mitotic Nuclear Envelope Disassembly 0.026 3 1 2 1 1 1 1 1 1 44 GO:0048856 Anatomical Structure Development 1 12 0.057 14 0.102 14 0.503 15 1 10 1 11 0.009 17 3E-06 22 1 12 1E-05 20 0.231 16 0.329 13 5272 GO:0048731 System Development 1 11 0.224 12 1 11 1 13 1 8 1 10 0.107 14 0.035 15 1 10 0.034 14 1 13 1 10 4482 GO:0048869 Cellular Developmental Process 1 8 0.461 11 0.735 11 1 12 0.025 13 1 6 1 11 7E-05 18 1 7 0.002 15 1 11 0.127 12 4112 GO:0030154 Cell Differentiation 1 8 1 10 1 10 1 12 0.336 11 1 6 1 11 2E-04 17 1 7 0.001 15 1 10 0.069 12 3859 GO:0001649 Osteoblast Differentiation 1 3 1 2 1 2 1 2 1 1 1 2 1 1 0.046 4 209 GO:0003012 Muscle System Process 1 1 1 1 1 2 1 1 1 3 1 1 1 2 1 2 1 1 0.031 5 398 GO:0012501 Programmed Cell Death 1 8 1 3 1 1 0.326 9 0.034 9 1 8 0.003 11 0.179 9 0.384 8 1 3 1 5 1 7 1921 GO:0097194 Execution Phase of Apoptosis 1 1 1 1 0.122 3 0.004 4 1 2 1 2 1 1 96 GO:0030262 Apoptotic Nuclear Changes 0.004 3 0.006 3 1 1 1 1 30 GO:0006309 Apoptotic DNA Fragmentation 7E-04 3 0.264 2 1 1 18 GO:0009607 Response to Biotic Stimulus 1 5 1 4 1 3 1 5 1 2 1 3 0.008 8 0.089 7 1 2 1 1 1 3 1 4 984 GO:0006974 Cellular Response to DNA Damage Stimulus 0.037 7 1 4 1 5 3E-05 9 1 3 1 5 1 1 1 4 1 3 0.5 6 0.007 7 811 GO:0006979 Response to Oxidative Stress 1 3 1 2 1 2 1 1 1 3 1 2 1 1 1 4 0.033 5 401 GO:0070887 Cellular Response to Chemical Stimulus 1 9 0.103 10 0.842 9 1 10 1 6 1 9 0.124 11 0.16 11 0.007 12 2E-06 16 1 9 0.003 12 2852 GO:0014070 Response to Organic Cyclic Compound 1 2 1 4 1 3 1 4 1 3 1 2 0.382 6 1 5 1 3 1E-04 9 1 4 1 4 890 GO:1901698 Response to Nitrogen Compound 1 1 1 3 1 4 1 5 1 5 1 6 0.019 8 1 2 1 3 1 1 1 5 1087 GO:1901700 Response to Oxygen-Containing Compound 1 3 1 5 1 6 1 6 1 4 1 5 0.006 10 0.058 9 1 6 0.021 9 1 4 0.086 8 1660 GO:0010038 Response to Metal Ion 1 1 0.014 5 1 3 1 2 1 1 1 1 1 2 1 2 1 2 1 2 1 2 349 GO:0006952 Defense Response 1 4 1 2 1 3 1 3 1 7 1 5 0.018 10 0.144 9 1 3 1 3 1 3 1 6 1867 GO:0002376 Immune System Process 1 6 1 3 1 4 1 6 0.002 12 1 6 0.069 11 0.003 13 1 5 1 7 1 6 1 8 2673 GO:0032606 Type I Interferon Production 1 1 1 1 0.006 4 0.013 4 1 2 1 1 1 1 127 GO:0007165 Signal Transduction 1 11 1 10 1 10 0.009 19 1 9 0.479 16 0.036 17 1E-04 21 1 9 0.009 17 1 13 0.244 14 5865 GO:0007154 Cell Communication 1 12 1 12 1 10 0.008 20 1 11 0.119 18 0.03 18 2E-05 23 1 10 0.001 19 0.215 18 0.165 15 6420 No. in Top 4% 63 49 50 63 49 62 58 60 53 52 66 49

Figure 5. GO term (Biological Process) enrichments of the 3rd-Z-4% cargoes. The 3rd-Z-4% cargoes were analyzed for GO term (term type, Biological Process) enrichment. The significantly enriched terms (p<0.05, cyan) in the 3rd-Z-4% cargoes of four or fewer NTRs were selected, and a representative term for each group of highly similar is presented with their p-values and the numbers (#) of cargoes annotated with them. Total No. denotes the number of proteins annotated with each term in the database. Related terms are bundled in the same color. This table was extracted from Figure 5 continued on next page

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Figure 5 continued Supplementary file 6B. All the GO terms annotated to the 3rd-Z-4% cargoes are listed in Supplementary file 7. The correspondence between each 3rd-Z-4% cargo and GO term is summarized in Supplementary file 9. For the 2nd-Z-15% cargoes, see Supplementary files 6A, 8, and 10. DOI: 10.7554/eLife.21184.013

Nearly twice as many GO terms were annotated to the 2nd-Z-15% cargoes. The correspondences between the 2nd-Z-15% cargoes of the 12 NTRs and selected representative GO terms enriched significantly for them are tabulated in Supplementary file 10. The excerpted list of terms enriched significantly for the cargoes of four or fewer NTRs contains terms partially different from those in Figures 5 and 6 (Supplementary file 6A), but many of the NTRs are still linked to terms similar to those of the 3rd-Z-4% cargoes. For example, Imp-b and Trn-SR are linked to terms related to cell or nuclear division by the 3rd-Z-4% cargoes (Figure 5) and to partially different terms that are still related to cell or nuclear division by the 2nd-Z-15% cargoes (Supplementary file 6A). Additionally, Imp-4 is linked to terms related to DNA structure regulation, DNA repair, and apoptosis in both lists. Similarly, most of the examined NTRs are linked in both lists to similar terms that are related to any of the following: chromatin organization, chromosome organization, DNA repair, ribosome biogenesis, protein modification, cell division, nuclear division, and apoptosis. Thus, we regard the 3rd-Z-4% list as a core table of the cargo roles. Naturally, the 2nd-Z-15% cargoes linked the NTRs to additional terms (Supplementary file 6A) as follows: Imp-b, -4, and Trn-SR are linked to DNA-dependent DNA replication; Trn-1 and Imp-7 are linked to gene silencing by RNA; Imp-b, -7, and -13 are linked to rRNA transcription; Imp-b and Trn-SR are linked to protein methylation; Trn-SR is linked to protein peptidyl-prolyl isomerization; Trn-2, Imp-4, and -8 are linked to circadian rhythm; Imp-b, -4, -13, and Trn-SR are linked to terms for CCs and MFs that are related to RNA polymerase (RNAP) II transcription; and subsets of the NTRs are linked to varying terms that are related to differentiation, development, and response. As an important result, we have illustrated the general framework of the division of roles among the NTRs for the first time, in which one NTR is linked to many BPs and conversely each broadly defined BP is supported by many NTRs, but each closely defined BP is supported by a restricted number of NTRs. One typical example is the allocation of mRNA processing factors (see below).

Allocation of mRNA processing factors to the NTRs Some of the GO terms related to mRNA processing were specifically linked to four or fewer NTRs by the 3rd-Z-4% and 2nd-Z-15% cargoes (Figure 5; Supplementary file 6A). However, many other terms related to mRNA processing were linked to more NTRs, and conversely, all the NTRs were implicated in mRNA processing. The 2nd- and 3rd-Z-rankings for the 12 NTRs included 275 and 242 proteins, respectively, that were annotated with mRNA processing (Supplementary file 6B and 6C). To see the allocation of these proteins to the NTRs, the ranks of these proteins are arranged in a table (Supplementary file 11A). The 2nd- and 3rd-Z-rankings revealed similar results. As summarized for the 2nd-Z-ranking (Figure 7), particular groups of the mRNA-processing factors are allocated to specific NTRs, showing that each NTR is linked to distinct reactions in mRNA processing: the proteins related to mRNA capping are allocated to Imp-b almost exclusively; hnRNP A0 is allocated to Trn-1, -2, Imp-4, -11, and others; hnRNP A1, A2B1, A3, D, F, H1–3, and M are allocated to Trn-1 and -2, and additionally Imp-9 and Exp-4; hnRNP U-like 1 are allocated to Imp-7, -8, and -9; SR-rich SFs are primarily allocated to Trn-SR and secondarily to Imp-7, -8, and -9; SFs 3A and B are allocated to Imp-4, -7, -8, -9, -11, and Exp-4; PQ-rich SF is allocated to Imp-4, -7, -8, and Exp-4; snRNP A–C is allocated to Imp-4, -7, -8, and Exp-4; exon junction complex (EJC) components are exclusively allocated to Imp-11 and -13; cleavage and polyadenylation specificity factor (CPSF) 1 is allocated to Imp-7 and -9; CPSF5 (NUDT21), 6, and 7 are less specifically allocated to other NTRs; general transcription factor IIF is allocated to Imp-b, -4, and Trn-SR; and RNAP II associating factors are allocated to Trn-SR and separately to other NTRs. Thus, the NTRs import distinctive subsets of mRNA processing factors. In the 2nd-Z-ranking, the SR-rich SFs were not allocated to Imp-5, but they were identified as the Imp-5 3rd-Z-4% cargoes. Thus, subsets of proteins involved in a broadly defined BP, for example, mRNA processing, are allocated to different NTRs, in a manner representative of role division among the NTRs.

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A Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Total Term ID Term Name p # p # p # p # p # p # p # p # p # p # p # p # No. GO:0008622 Epsilon DNA Polymerase Complex 0.024 2 5 GO:0044452 Nucleolar Part 1 2 1 1 0.033 3 0.076 3 1 1 0.076 3 1 2 1 1 3E-04 4 60 GO:0015030 Cajal Body 1 1 6E-04 4 6E-04 4 1 1 0.024 3 1 2 0.019 3 52 GO:0042382 Paraspeckles 1 1 1 1 1 1 1 1 1 1 2E-05 3 6 GO:0034399 Nuclear Periphery 1 1 1 2 1 1 1 1 0.249 3 1 1 1 1 0.31 3 1 1 1 2 0.006 4 122 GO:0005635 Nuclear Envelope 2E-05 8 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 3 1 1 0.172 5 1 2 410 GO:0000785 Chromatin 0.012 6 0.724 4 1 1 1 2 1E-04 7 1 3 1 3 1 2 0.073 5 1 2 1 3 0.003 6 442 GO:0000791 Euchromatin 1 1 0.005 3 1 1 31 GO:0098687 Chromosomal Region 1 3 1 2 2E-06 8 1 1 1 3 1 3 1 1 1 1 1 3 1 3 301 GO:0000793 Condensed Chromosome 1 3 1 1 0.004 5 0.032 4 1 1 0.067 4 1 1 191 GO:0000776 Kinetochore 0.015 4 1 1 1 1 1 2 1 2 116 GO:0005819 Spindle 1 3 1 1 4E-05 7 1 1 0.455 4 1 2 1 2 1 2 279 GO:0005813 Centrosome 1 1 1 1 0.001 7 1 2 1 2 1 1 1 2 476 GO:0090575 RNA Polymerase II Transcription Factor 0.006 4 1 2 1 2 1 2 1 1 1 1 0.008 4 1 2 96 Complex GO:0016602 CCAAT-Binding Factor Complex 1 1 1 1 0.014 2 4 GO:0005845 mRNA Cap Binding Complex 1E-06 4 12 GO:0005732 Small Nucleolar Ribonucleoprotein Complex 1 1 1 1 1 1 1 1 0.441 2 1 1 0.001 3 20 GO:0030532 Small Nuclear Ribonucleoprotein Complex 1 1 1 2 1E-19 11 0.054 3 4E-06 5 1 1 60 GO:0097525 Spliceosomal snRNP Complex 1 1 1 2 3E-20 11 0.037 3 2E-06 5 1 1 53 GO:0005684 U2-Type Spliceosomal Complex 0.008 3 5E-10 6 0.006 3 1 1 1 1 29 GO:0089701 U2AF 1 1 0.002 2 0.002 2 0.001 2 1 1 2 GO:0005849 mRNA Cleavage Factor Complex 1 1 1 1 1 1 3E-04 3 14 GO:0044391 Ribosomal Subunit 0.019 4 1 1 1 1 1 3 3E-09 8 0.727 3 1 2 162 GO:0031428 Box C/D snoRNP Complex 1 1 1 1 1 1 1 1 0.018 2 6 GO:0005856 Cytoskeleton 0.293 9 1 5 1 3 2E-04 13 1 5 1 7 1 4 1 6 1 5 0.001 11 0.014 11 1 4 1949 GO:0010494 Cytoplasmic Stress Granule 1 1 1 1 1 1 1 2 1 1 0.008 3 32 GO:0044445 Cytosolic Part 0.048 4 1 2 1 1 1 3 2E-08 8 1 3 1 2 205 GO:0005739 Mitochondrion 0.622 8 1 5 1 4 1 4 0.102 8 1 5 9E-04 11 1E-05 13 3E-04 11 0.003 10 0.966 8 0.102 8 1699 GO:0005783 Endoplasmic Reticulum 1 2 1 3 1 4 1 1 1 5 1 3 1 6 8E-04 11 1 1 1 2 1 4 1 5 1623 GO:0005768 Endosome 1 1 1 1 1 2 1 4 1 3 1 4 1 3 2E-08 12 1 1 1 2 1 1 1 3 779 GO:0005794 Golgi Apparatus 1 2 1 2 1 3 1 5 1 2 1 2 2E-04 11 1 3 1 3 1 5 1 2 1436 GO:0031410 Cytoplasmic Vesicle 1 1 1 2 1 2 1 4 1 5 1 3 1 4 3E-07 13 1 4 1 2 1 1 1 5 1232 GO:0005925 Focal Adhesion 1 2 1 1 1 2 1 1 1 4 1 1 0.004 6 0.002 6 0.039 5 0.008 6 1 2 383 GO:0005905 Coated Pit 1 1 1 2 1 1 1 2 1 1 1 1 1 1 0.033 3 62 GO:0043209 Myelin Sheath 1 1 1 2 1 3 0.047 4 1 2 1 2 0.032 4 1 1 1 1 175 No. in Top 4% 63 49 50 63 49 62 58 60 53 52 66 49 B Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 Total Term ID Term Name p # p # p # p # p # p # p # p # p # p # p # p # No. GO:0034061 DNA Polymerase Activity 0.008 3 1 1 1 2 30 GO:0003690 Double-Stranded DNA Binding 0.023 7 1 4 1 4 1 2 0.62 5 1 5 0.153 6 1 3 1 4 1 2 0.62 5 752 GO:0003697 Single-Stranded DNA Binding 1 1 0.001 4 1 2 1 1 1 2 0.164 3 1 2 1 1 1 2 1 2 88 GO:0098847 Sequence-Specific Single Stranded DNA 0.039 2 0.046 2 1 1 9 Binding GO:0008301 DNA Binding, Bending 0.001 3 0.002 3 20 GO:0043566 Structure-Specific DNA Binding 0.289 3 1 1 1 1 0.002 4 0.226 3 0.162 3 1 1 98 GO:0000217 DNA Secondary Structure Binding 7E-04 3 0.001 3 1 1 18 GO:0000400 Four-Way Junction DNA Binding 5E-05 3 9E-05 3 1 1 8 GO:0097100 Supercoiled DNA Binding 0.007 2 0.011 2 1 1 4 GO:0003682 Chromatin Binding 8E-11 12 1 3 1 2 1 4 0.061 5 1 2 1 2 1 3 1E-05 8 1 1 1 4 0.924 4 457 GO:0042162 Telomeric DNA Binding 0.003 3 0.004 3 1 1 30 GO:0001047 Core Promoter Binding 1 3 1 1 1 2 1 2 1 2 1 2 1 2 0.91 3 0.019 4 1 2 0.514 3 156 GO:0000988 Transcription Factor Activity, Protein Binding 3E-04 8 0.012 6 0.227 5 0.006 7 1 1 1 2 1 2 1 2 1 4 1 3 1 4 1 2 587 GO:0003727 Single-Stranded RNA Binding 1 2 2E-08 6 4E-04 4 1 1 1 1 1 2 1 1 1 2 1 2 1 2 62 GO:0036002 Pre-mRNA Binding 1 1 1 1 0.004 3 1 1 1 1 1 1 1 1 1 1 1 1 23 GO:0071208 Histone Pre-mRNA DCP Binding 0.011 2 4 GO:0003730 mRNA 3'-UTR Binding 1 1 0.015 3 0.018 3 1 1 1 1 49 GO:0017091 AU-rich Element Binding 0.001 3 0.002 3 1 1 0.432 2 1 1 23 GO:0017069 snRNA Binding 1 1 1 1 1 1 1 1 3E-07 5 1 1 1 1 1 1 1 1 34 GO:0035614 snRNA Stem-Loop Binding 0.002 2 2 GO:0004004 ATP-Dependent RNA Helicase Activity 2E-07 6 1 1 0.102 3 0.001 4 0.05 3 1 2 1 1 1 2 66 GO:0030515 snoRNA Binding 1 1 1 1 1 1 0.005 3 1 1 0.471 2 0.323 2 24 GO:0051082 Unfolded Protein Binding 1 1 0.009 4 1 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 102 GO:0019789 SUMO Transferase Activity 4E-04 3 11 GO:0019901 Protein Kinase Binding 1 1 1 2 1 3 0.046 6 1 2 1 3 1 2 1 3 1 1 1 3 1 4 1 3 543 GO:0003988 Acetyl-CoA C-Acyltransferase Activity 0.021 2 6 GO:0016887 ATPase Activity 1 4 1 1 1 1 0.008 6 1 1 1 3 1 2 1E-05 8 1 3 1 3 1 1 1 3 402 GO:0003924 GTPase Activity 1 3 1 1 1 1 1 3 0.126 4 2E-09 9 1 2 1 2 225 No. in Top 4% 63 49 50 63 49 62 58 60 53 52 66 49

Figure 6. GO term (Cellular Component and Molecular Function) enrichments of the 3rd-Z-4% cargoes. The 3rd-Z-4% cargoes were analyzed, and the results are presented in a format similar to that of Figure 5.(A) Term type, Cellular Component. (B) Term type, Molecular Function. These tables were extracted from Supplementary file 6B. All the GO terms annotated to the 3rd-Z-4% cargoes are listed in Supplementary file 7. The correspondence between each 3rd-Z-4% cargo and GO term is summarized in Supplementary file 9. For the 2nd-Z-15% cargoes, see Supplementary file 6A, 8, and 10. DOI: 10.7554/eLife.21184.014

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Rank by 2nd Z-score Accession Major Feature Gene Name Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 O43148 mRNA Capping RNMT 2 1432 1420 1906 358 327 1487 1066 687 1560 598 447 Q09161 Cap-Binding NCBP1 25 327 1037 1373 751 1466 906 1220 655 1248 594 1075 P52298 NCBP2 19 1416 1529 865 1510 602 Q13151 hnRNP HNRNPA0 247 11 13 145 50 137 92 422 105 7 558 82 P09651 HNRNPA1 245 7 7 285 364 776 323 438 171 773 641 177 P22626 HNRNPA2B1 301 16 19 382 555 902 336 420 160 711 531 123 P51991 HNRNPA3 634 13 14 531 260 823 165 163 182 405 987 210 P07910 HNRNPC 496 857 259 386 324 1140 337 570 117 563 676 229 Q14103 HNRNPD 851 8 8 566 623 1300 565 938 709 1237 1512 633 P52597 HNRNPF 1678 95 52 1702 382 211 268 230 608 1289 1251 304 P31943 HNRNPH1 438 34 40 948 171 723 233 325 243 628 646 202 P55795 HNRNPH2 741 41 42 1452 1126 534 363 280 302 802 573 208 P31942 HNRNPH3 373 167 85 753 708 686 221 177 177 394 404 184 P61978 HNRNPK 509 1130 331 351 611 919 459 569 318 982 580 315 P14866 HNRNPL 453 491 125 634 154 477 532 621 148 999 921 236 Q8WVV9 HNRPLL 145 1397 1430 1331 1281 774 320 542 479 1018 1056 212 P52272 HNRNPM 403 4 5 294 532 754 618 723 216 333 777 292 O60506 SYNCRIP 770 866 483 1123 503 508 435 529 575 808 628 693 O43390 HNRNPR 471 1421 133 374 1269 598 463 719 293 1236 674 388 Q00839 HNRNPU 123 837 205 395 415 1025 759 1247 475 1133 645 556 Q9BUJ2 HNRNPUL1 572 1600 1586 1503 354 1382 47 50 9 1253 1323 100 Q8IYB3SR-Rich SRRM1 1440 1040 1977 863 1842 276 6 723 Q9UQ35Splicing Factor SRRM2 202 203 147 3 1412 1463 249 54 151 803 1872 355 Q07955 SRSF1 1617 328 113 55 984 1159 105 84 238 490 786 145 Q01130 SRSF2 133 234 143 8 156 628 68 85 152 373 83 164 P84103 SRSF3 274 294 254 90 243 379 110 118 49 236 357 92 Q13243 SRSF5 1002 213 378 524 538 833 457 659 212 1504 644 379 Q13247 SRSF6 1964 212 279 16 740 537 288 446 124 1091 1728 287 Q16629 SRSF7 377 553 592 26 200 380 86 120 85 224 232 79 Q9BRL6 SRSF8 115 738 1244 19 369 34 Q05519 SRSF11 1194 342 162 1764 221 1199 81 81 93 338 1576 185 Q15459Splicing Factor SF3A1 1621 442 366 1667 118 982 206 228 190 184 1313 150 Q154283A, B SF3A2 1623 402 320 1644 126 782 177 209 202 106 1414 133 Q12874 SF3A3 1650 375 291 1674 114 1021 222 243 231 227 1367 160 O75533 SF3B1 1660 343 273 1542 81 862 211 147 188 56 1326 84 Q13435 SF3B2 1638 432 276 1522 143 1000 282 206 178 214 1491 151 Q15393 SF3B3 1604 322 288 1516 172 999 316 352 192 71 1191 207 Q15427 SF3B4 1552 458 214 1566 215 1029 311 217 180 126 843 217 Q9BWJ5 SF3B5 1634 418 247 1478 265 1077 217 207 229 77 1317 188 Q9Y3B4 SF3B14 1663 336 246 1685 43 912 181 188 330 148 1483 131 Q01081Splicing Factor U2AF1 190 154 201 202 329 175 99 148 48 177 1476 66 P26368U2AF U2AF2 261 360 342 1293 220 218 111 128 76 286 1107 106 P23246 PQ-Rich SF SFPQ 111 219 1209 1890 31 1831 74 24 547 1542 143 42 P09012snRNP A-F SNRPA 1030 444 400 218 459 1063 117 142 218 415 1887 361 P09661 SNRPA1 1463 338 316 1664 112 976 207 231 156 107 1475 141 P14678 SNRPB 764 333 222 1540 238 795 228 143 210 226 736 219 P08579 SNRPB2 1422 311 257 1603 84 1050 183 216 144 268 1510 85 P09234 SNRPC 354 555 393 915 411 825 130 170 272 310 710 91 P62314 SNRPD1 743 300 241 1271 166 896 359 441 196 481 1602 364 P62316 SNRPD2 1199 604 238 1646 284 1308 352 493 311 342 1084 227 P62318 SNRPD3 843 384 226 1523 549 861 260 162 217 282 631 256 P62304 SNRPE 1512 357 203 1399 230 948 285 293 241 396 1458 431 P62306 SNRPF 584 426 466 893 73 1191 171 266 223 240 562 329 Q9Y5S9Exon Junction RBM8A 497 517 505 976 783 1857 1614 1052 281 109 9 570 P38919Complex EIF4A3 629 289 392 256 394 998 535 852 490 24 737 681 P61326 MAGOH 1643 388 329 1183 484 1414 929 975 402 841 8 450 Q10570Cleavage and CPSF1 1029 690 1105 1481 231 1950 48 1236 56 1586 1837 114 Q9P2I0Polyadenylation CPSF2 825 578 351 309 1434 1326 917 1541 530 99 1856 1730 Q9UKF6Specificity CPSF3 1104 1348 274 1367 1270 1666 1358 1664 663 1639 1359 O43809Factor NUDT21 415 335 99 157 593 253 254 165 154 590 1478 254 Q16630 CPSF6 281 647 368 125 201 289 125 96 121 738 687 103 Q8N684 CPSF7 187 521 191 62 421 145 158 208 89 138 1136 62 P35269General GTF2F1 35 587 192 68 64 685 224 846 416 466 210 309 P13984Transcription GTF2F2 61 636 249 134 229 964 741 811 236 837 510 472 Q8N7H5RNA Pol II PAF1 1901 886 1304 14 579 1478 1224 827 1657 33 Q6P1J9Associating CDC73 1478 266 542 206 992 1355 484 1227 329 1635 1829 193 Q6PD62Factor CTR9 106 1398 91 189 29 159 79 760 1509 324 Q8WVC0 LEO1 1248 1271 792 272 624 1570 574 1601 187 1546 1277 404 P18615 NELFE 911 1482 1117 123 55 737 757 1317 487 1199 1891 882 Color Scale: Top 4% 4–8% 8–15%

Figure 7. mRNA processing factors in the 2nd-Z-rankings. The ranks of the mRNA processing factors in the 2nd-Z-rankings of the 12 NTRs are presented. The color scale is set by percentile rank as indicated. The 2nd-Z-rankings of the 12 NTRs include 275 proteins in total that are annotated with mRNA processing in GO. Of these, 69 were selected and are presented. For other factors and the 3rd-Z-rankings, see Supplementary file 11A. DOI: 10.7554/eLife.21184.015

Allocation of RPs to the NTRs RPs migrate into the nuclei for ribosome assembly, but the NTRs responsible for import have been determined for only a few of RPs (Chook and Su¨el, 2011). The 3rd-Z-4% cargoes include 15 RPs (Supplementary files 1, 5C, and 11B). To see the allocations of all the RPs to the NTRs, the ranks of

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 14 of 31 Tools and resources Biochemistry Cell Biology the RPs in the 2nd-Z-rankings are arranged in a table (Figure 8). Because the +NTR/Ctl values were obtained for most of the RPs in the three SILAC-Tp replicates, the second Z-scores are the median Z-scores in most cases, and they should fairly reflect the import efficiencies. Half of the RPs are included in the 2nd-Z-15% cargoes of one to five NTRs, and most of the RPs are ranked in the top 30% in the 2nd-Z-rankings of additional NTRs. Surprisingly, most RPs, especially the 60S subunit proteins, are ranked in the top 50% of most of the 2nd-Z-rankings, and few RPs are ranked lower. These findings imply that most of the RPs are allocated to multiple NTRs, but the import efficiencies vary depending on the NTR. Indeed, several RPs are reported cargoes of multiple NTRs (Ja¨kel and Go¨r- lich, 1998; Ja¨kel et al., 2002). Imp-7, -8, and -9 primarily import RPs, Imp-11 and Exp-4 secondarily import RPs, and all other NTRs also contribute to the import of RPs to some extent. Among the highly homologous NTR pairs, Trn-2 and Imp-8 import RPs more efficiently than Trn-1 and Imp-7, respectively, which indicates that RP import is one of the roles shared unequally by similar NTRs. This differentiation is clearer in the 3rd-Z-rankings (Supplementary file 11B).

Allocation of transcription factors to the NTRs Sequence-specific DNA-binding transcription factors (annotated with ‘transcription factor activity, sequence specific DNA binding’ in GO) play pivotal roles in many cellular processes, but they are not significantly enriched in the 3rd-Z-4% cargoes of any NTR (Supplementary file 6B). Transcription cofactors (annotated with ‘transcription factor activity, protein binding’), which may engage in gene- specific transcription, are significantly enriched in the 3rd-Z-4% cargoes of only three NTRs (Figure 6B; Supplementary file 6B). Nonetheless, transcription factors (sequence-specific DNA binding) are enriched in the Imp-b 2nd-Z-15% cargoes, and cofactors (protein binding) are enriched in the 2nd-Z-15% cargoes of 10 NTRs (Supplementary file 6C). Additionally, some transcription factors and cofactors are included in the 2nd-Z-15% cargoes, albeit not enriched. Thus, the 2nd-Z-15% cargoes of each NTR include 17 to 36 transcription factors or cofactors as listed at the bottom of Supplementary file 11D. We performed GO analyses (term type, BP) for these transcription factors and cofactors (Supplementary file 11C and 11D). The annotated terms may reflect both direct transcription regulation activities and indirect effects via transcription. The proteins annotated with histone modification are enriched in the Imp-b and -13 cargoes, and the term may reflect their direct functions. The cargoes of several NTRs annotated with varying types of nuclear receptor signaling may act as cofactors in receptor-regulated transcription. In contrast, many of the transcription factors and cofactors identified as cargoes are annotated differently with various terms related to cell prolif- eration, development, rhythmic processes, or apoptosis and may act on these processes via transcriptional regulation. Thus, the NTRs import transcription factors and cofactors that work in distinct cellular processes.

Characterizations of the cargoes of individual NTRs The GO analyses elucidated the characteristics of the NTR-specific cargoes, but the terms are annotated to not only the central players but also many indirect participants in BPs. Here, we primarily discuss the roles of the notable 3rd-Z-4% cargoes of each NTR and supplement this information with references to the 2nd-Z-15% cargoes. To make our points clear, we classified the 3rd-Z-4% and 2nd- Z-15% cargoes by their characteristics and their allocations to each NTR are presented in Supplementary file 5C and 5E. We describe the features of the Imp-13 and Trn-SR cargoes first, because it includes the discussion on an export cargo or SR-domains. Biological functions linked to NTRs by the natures of their cargoes need to be verified by further experiments.

Imp-13 cargoes Several Imp-13 cargoes have previously been reported, and our SILAC-Tp clearly reproduced the reported import specificities. Nuclear transcription factor Y subunits b (NFYB) and g (NFYC) have been reported to be Imp-13 specific cargoes, whereas subunit a (NFYA), which has a BIB-like sequence, has been reported to bind to multiple NTRs (Kahle et al., 2005). NFYB is ranked first in both the Imp-13 2nd- and 3rd-Z-rankings, and NFYC is a 2nd-Z-15% cargo (Figure 3; Supplementary files 1 and 3). Additionally, we identified NFYA as a cargo of multiple NTRs. Inter- estingly, a subunit of the general transcription factor TFIIA (GTF2A2) that interacts with NFYA (Rolland et al., 2014) is also a highly ranked Imp-13 3rd-Z-4% cargo. We could not identify the Imp-

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Rank by 2nd Z-score Accession Gene Name Imp- Trn-1 Trn-2 Trn-SR Imp-4 Imp-5 Imp-7 Imp-8 Imp-9 Imp-11 Imp-13 Exp-4 P08865 RPSA 1112 1181 580 1143 442 599 568 436 520 514 805 640 P15880 RPS2 1279 860 840 992 1095 859 633 488 481 565 740 594 P23396 RPS3 1266 1058 645 1042 962 657 537 418 512 372 973 494 P61247 RPS3A 1127 1047 859 1096 672 745 548 413 351 455 827 475 P62701 RPS4X 1197 911 835 982 925 624 474 394 422 558 800 564 P46782 RPS5 1094 967 678 1095 725 769 478 252 338 502 798 458 P62753 RPS6 673 902 456 1173 590 829 507 502 395 522 791 369 P62081 RPS7 1103 1031 907 1127 889 655 511 431 81 588 747 560 P62241 RPS8 1161 1083 871 1020 927 691 530 464 424 503 691 489 P46781 RPS9 1147 1016 699 963 704 626 392 404 366 523 880 535 P46783 RPS10 924 1105 521 669 675 703 420 335 328 253 662 490 P62280 RPS11 1170 1086 743 735 842 555 490 390 369 587 751 448 P25398 RPS12 1110 1166 727 1033 1029 752 418 326 411 623 619 482 P62277 RPS13 1087 830 888 1140 812 693 516 429 420 521 916 456 P62263 RPS14 1089 1203 936 1248 1075 881 319 382 418 657 689 623 P62841 RPS15 1373 908 691 997 1002 487 510 449 583 953 936 612 P62244 RPS15A 1037 1263 862 1014 878 651 503 432 436 329 1020 546 P62249 RPS16 1045 1041 763 966 957 588 517 369 371 583 821 653

40S Subunit P0CW22 RPS17L 1210 1158 709 802 784 512 520 447 361 403 1018 441 P62269 RPS18 981 1089 674 869 911 583 467 391 333 729 977 521 P39019 RPS19 1122 1099 937 993 888 594 461 384 360 285 851 506 P60866 RPS20 1123 968 858 736 995 472 424 226 365 293 701 544 P63220 RPS21 1215 988 954 1036 718 653 704 340 444 549 502 614 P62266 RPS23 1219 957 776 785 756 475 305 297 348 516 634 345 P62847 RPS24 1017 1056 899 1073 890 652 496 409 290 484 779 486 P62851 RPS25 1111 1211 1052 925 563 812 450 313 525 428 1066 410 P62854 RPS26 975 1400 1428 885 1000 916 428 250 371 1242 P42677 RPS27 1051 997 684 811 616 595 514 405 428 610 446 631 P62979 RPS27A 710 451 1108 966 668 828 350 483 265 489 577 P62857 RPS28 828 1106 802 809 917 596 557 367 401 598 726 391 P62273 RPS29 1256 1064 1068 873 916 722 519 318 536 1056 702 365 P62861 FAU(RPS30) 1265 981 675 1487 908 990 528 320 403 150 695 407 P05388 RPLP0 1152 1066 662 1028 1146 711 690 821 564 731 1069 520 P05386 RPLP1 526 732 650 1030 373 913 817 678 594 416 323 503 P05387 RPLP2 908 733 476 766 929 666 575 481 686 260 302 401 P39023 RPL3 830 665 524 1047 455 800 332 160 184 366 824 161 P36578 RPL4 832 563 410 1247 633 891 317 302 207 459 758 246 P46777 RPL5 873 638 566 841 853 1053 830 843 624 612 889 778 Q02878 RPL6 701 562 478 1199 601 815 389 331 306 689 578 340 P18124 RPL7 816 618 457 1057 739 789 354 227 137 157 716 305 Q6DKI1 RPL7L1 1842 1483 41 P62424 RPL7A 929 775 582 1230 404 639 353 237 262 448 743 180 P62917 RPL8 728 731 481 1266 504 773 333 246 153 234 757 303 P32969 RPL9 1153 662 347 1334 368 806 318 195 265 595 870 291 P27635 RPL10 960 789 668 1177 974 809 399 311 263 489 712 420 P62906 RPL10A 808 621 360 1227 461 787 313 241 147 471 769 251 P62913 RPL11 793 712 491 1326 1123 710 497 40 201 375 401 377 P30050 RPL12 863 763 614 1208 305 814 338 321 254 314 845 286 P26373 RPL13 836 530 492 1197 275 775 244 185 189 323 759 247 P40429 RPL13A 737 940 375 1203 943 864 239 362 228 305 656 398 P50914 RPL14 814 1136 561 1204 290 749 299 154 240 573 897 362 P61313 RPL15 801 771 432 1246 556 820 388 244 205 360 841 353 P18621 RPL17 799 1004 706 1170 610 350 308 315 209 518 597 166 Q07020 RPL18 758 760 538 1229 369 971 248 368 269 422 754 314 Q02543 RPL18A 847 484 551 1400 1285 830 437 328 232 304 605 289 P84098 RPL19 1109 1118 651 1243 406 695 502 301 384 176 975 367 P46778 RPL21 818 883 507 1161 647 836 382 319 247 607 509 139 P35268 RPL22 729 714 450 474 515 306 52 42 347 1021 398 376 60S Subunit P62829 RPL23 817 703 535 897 668 842 536 448 323 718 836 514 P62750 RPL23A 1092 625 417 1146 844 807 162 222 165 458 753 358 P83731 RPL24 1035 963 718 1141 643 743 421 307 299 139 1001 381 P61254 RPL26 650 821 523 1160 664 636 192 229 320 547 649 279 Q9UNX3 RPL26L1 1337 1693 454 567 122 P61353 RPL27 705 654 550 1158 614 925 301 192 326 430 941 216 P46776 RPL27A 792 615 477 1151 295 901 306 274 211 105 599 182 P46779 RPL28 666 566 412 1325 203 603 409 346 342 806 555 198 P47914 RPL29 982 682 598 1219 660 660 303 235 346 615 847 294 P62888 RPL30 621 659 570 1252 760 570 273 136 173 370 808 220 P62899 RPL31 543 1026 590 1142 1104 811 567 286 376 379 568 336 P62910 RPL32 885 583 416 1324 301 926 381 199 252 1055 910 242 P49207 RPL34 1135 1410 350 1285 597 546 278 123 305 223 658 260 P42766 RPL35 731 508 356 1058 518 593 175 168 104 67 454 128 P18077 RPL35A 951 614 772 1366 814 840 385 219 193 255 813 102 Q9Y3U8 RPL36 1157 845 317 1231 125 961 414 395 128 239 823 230 P83881 RPL36A 1238 382 295 1511 Q969Q0 RPL36AL 1347 522 937 211 609 601 489 168 P61927 RPL37 309 1012 458 1638 130 474 P61513 RPL37A 1142 827 526 940 613 707 551 238 237 562 1075 162 P63173 RPL38 780 1052 409 1046 387 623 280 210 266 555 586 264

Color Scale: Top 15% 15-30% 30-50% >50%

Figure 8. Ribosomal proteins in the 2nd-Z-rankings. The ranks of the ribosomal proteins in the 2nd-Z-rankings of the 12 NTRs are presented. The color scale is set by the percentile rank as indicated. For the 3rd-Z-rankings, see Supplementary file 11B. DOI: 10.7554/eLife.21184.016

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 16 of 31 Tools and resources Biochemistry Cell Biology 13 reported cargo glucocorticoid receptor (Tao et al., 2006) in our MS, but proteins that may interact with nuclear receptors, e.g., thyroid hormone receptor-associated protein 3 (THRAP3) (Ito et al., 1999), RNA-binding protein 14 (RBM14) (Iwasaki et al., 2001), and transcription activator BRG1 (SMARCA4) (Dai et al., 2008), were identified as Imp-13 2nd-Z-15% cargoes. THRAP3 interacts with EJC (Lee et al., 2010), whose subunits, RNA-binding protein RBM8A and mago nashi homolog MAGOH, are well-characterized Imp-13 cargoes (Mingot et al., 2001). RBM8A and MAGOH are highly ranked Imp-13 3rd-Z-4% cargoes, which were not identified as cargoes of the other NTRs with the exception of Trn-1. However, another EJC subunit, that is, translation initiation factor 4A-III (EIF4A3) (Shibuya et al., 2004), was identified as an Imp-11 3rd-Z-4% cargo. Thus, the EJC subunits are imported through different pathways. A well-characterized Imp-13 cargo, SUMO-conjugating enzyme UBC9 (UBE2I) (Mingot et al., 2001), was identified as a 3rd-Z-4% cargo, and SUMO2 and SUMO3 were also identified as 3rd-Z-4% cargoes. Components of the chromatin accessibility complex CHRAC15 (CHRAC1) and DNA polymerase e subunit 3 (POLE3) are also Imp-13 reported cargoes (Walker et al., 2009), and they are highly ranked 3rd-Z-4% cargoes. In the GO analysis, Imp-13 was linked to chromatin modification by the 2nd-Z-15% cargoes (Supplementary file 6A). Nucleolar complex protein 2 homolog (NOC2L) is ranked 10th in both the second and third Z-scores, and lysine-specific demethylase 2A (KDM2A) is ranked second in the second Z-score. The Imp-13 2nd-Z- 15% cargoes include many actin-related proteins that are involved in chromatin remodeling and transcription (Oma and Harata, 2011; Yoo et al., 2007). Surprisingly, a reported Imp-13 export cargo eIF1A (EIF1AX; Mingot et al., 2001) was identified as a 2nd-Z-15% cargo (ranked 84th and 164th by the second and third Z-score, respectively; Supplementary files 1 and 3). If a protein endogenous to the permeabilized cell nuclei is exported

preferentially in the +NTR in vitro transport reaction, the (L/H+NTR)/(L/HCtl) value will be raised and the protein will be ranked high. However, it cannot be generalized because we have only one example. Most of the highly ranked Imp-13 cargoes must be import cargoes, because in all the bead halo assays where the cargoes bound to Imp-13 RanGTP inhibited the binding (Supplementary file 2).

Trn-SR cargoes The reported Trn-SR cargoes include SR-rich splicing factors (SFs) that coordinate transcription elongation, mRNA splicing, and mRNA export (Zhong et al., 2009). Here, we found that proteins engag- ing in these processes are also Trn-SR cargoes. The Trn-SR 3rd-Z-4% cargoes include the RNA polymerase (RNAP) II elongation factors NELFE and PAF1 (a subunit of the Paf1 complex, PAF1C), DDX and DHX family RNA helicases, and the THO complex subunit THOC1 as well as SR-rich SFs. The 2nd-Z-15% cargoes additionally include PAF1C subunits CTR9, CDC73, and LEO, FACT complex subunits SSRP1 and SPT16, additional DDX and DHX family helicases, and THOC6 and THOC3 (Supplementary file 5C and 5E). Trn-SR bound to NELFE, CDC73, DDX5, and DDX27 in the bead halo assays (Figure 3, Supplementary files 1, 2, and 3). Peptidyl-prolyl cis-trans isomerases, which are contained in human spliceosomes (Wahl et al., 2009), were also identified as 3rd-Z-4% and 2nd- Z-15% cargoes. DnaJ homologs were also identified as 3rd-Z-4% and 2nd-Z-15% cargoes, although the spliceosome component DNAJC8 (Zhou et al., 2002) was not. The 3rd-Z-4% cargoes also include proteins related to nuclear division or chromosome segregation, the Ser/Thr protein kinase PLK1, dual specificity protein kinase TTK, G2/M-specific cyclin-B1 (CCNB1), cyclin-dependent kinase (CDK) 2, protein FAM83D, and dynein 1 light intermediate chain 1 (DYNC1LI1) in addition to proteins related to histone acetylation or deacetylation including histone deacetylase complex subunit SAP18 and SAGA-associated factor 29 homolog CCDC101. Indeed, in the bead halo assays, Trn-SR bound to SAP18 and CCDC101 (Figure 3; Supplementary files 1, 2, and 3). Additionally, the 2nd- Z-15% cargoes include many proteins that are related to nucleosome or chromatin regulation. Thus, the Trn-SR cargoes are involved in chromosome regulation in addition to the coordination of transcription elongation, mRNA splicing, and mRNA export. Surprisingly, SR-rich SFs, which have been assumed to be Trn-SR-specific cargoes, were also identified as cargoes of other NTRs (Figure 7). To determine the allocation of the other SR-domain proteins to the NTRs, we here analyzed the distribution of SRSRSR hexa-peptide sequences in the 3rd- Z-4% cargoes (Supplementary file 11E). Imp-5, -7, -8, and Exp-4 as well as Trn-SR may be the specific NTRs for proteins with the hexa-peptide, most of which are nuclear proteins. The hexa-peptide- containing proteins other than the SR-rich SFs are primarily included in the Imp-5 and Exp-4 cargoes.

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 17 of 31 Tools and resources Biochemistry Cell Biology Imp-b cargoes The Imp-b cargoes play roles in DNA synthesis and repair and chromatin regulation. The Imp-b 3rd- Z-4% cargoes include DNA polymerase d subunits (POLD2 and 3) and mismatch repair endonuclease PMS2 (Supplementary file 5C). Additionally, the Imp-b 2nd-Z-15% cargoes include PCNA-associated factor KIAA0101 and DNA-(apurinic or apyrimidinic site) lyase (APEX1) (Supplementary file 5E). These proteins act in DNA synthesis or repair. The notable 3rd-Z-4% cargoes related to chromatin regulation include high-mobility group (HMG) proteins, histone acetyltransferase complex NuA4 subunit MRGBP, SWI/SNF-related regulator of chromatin SMARCE1, Spindlin-1 (SPIN1), chromodo- main-helicase CHD8, and lymphoid-specific helicase HELLS. Additionally, the 2nd-Z-15% cargoes include the NuA4 subunit MORF4L2, SWI/SNF complex subunit SMARCC2, chromatin assembly factor 1 subunit CHAF1B, polycomb protein EED, and sister chromatid cohesion protein PDS5B. Chro- matin remodeling by some of these factors is closely related to transcription. The 3rd-Z-4% cargoes include general transcription factor TFIIF (GTF2F1 and 2), TFIIH subunit MAT1, and TBP-associating factor TAF15, and the 2nd-Z-15% cargoes include TFIIH subunit cyclin-H (CCNH) and mediator complex subunit MED15. The sequence-specific transcription factors and cofactors are described above. mRNA capping factors are Imp-b cargoes as described. Thus, many Imp-b cargoes are related to the initial stage of gene expression.

Trn-1 and -2 cargoes The transcription factor ATF1 was ranked first in both the 2nd- and 3rd-Z-rankings of the Trn-1 and - 2 but was ranked low for the other NTRs (Figure 3; Supplementary files 3 and 5). As described, many of the cargoes that ranked higher in the Trn-1 and -2 2nd- and 3rd-Z-rankings (e.g. hnRNPs) are shared by Trn-1 and -2, but RPs are included only in the Trn-2 3rd-Z-4% cargoes. Additional divergences can be observed between their 2nd-Z-15% cargoes. As expected, their cargoes include many mRNA processing factors, but among them snRNPs are preferentially included in the Trn-2 2nd-Z-15% cargoes (Figure 7). Actin and actin-related proteins (ARPs), which play roles in chromatin remodeling and transcription (Visa and Percipalle, 2010; Yoo et al., 2007), proteins related to nuclear division, and tRNA ligases are preferentially Trn-1 cargoes, whereas proteins related to DNA repair and HMG proteins are preferentially Trn-2 cargoes (Supplementary file 5E).

Imp-4 cargoes In the GO analysis, Imp-4 was linked to DNA metabolic processes, chromosome organization, and related terms (Figures 5 and 6). Consistently, replication factor C subunit 5 (RFC5) and HMG proteins are Imp-4 3rd-Z-4% cargoes, and the 2nd-Z-15% cargoes include DNA polymerase a subunit POLA1, DNA ligase I (LIG1), DNA topoisomerase I (TOP1), SWI/SNF complex subunit SMARCC2, the SWI/SNF-related chromatin regulator SMARCA5, nucleosome remodeling factor subunit BPTF, and FACT complex subunit SPT16 (Supplementary file 5C and 5E). The participation of the Imp-4 cargoes in chromatin organization is supported by a report that Imp-4 binds to the histone chaperon complex (Tagami et al., 2004), although the subunits were not identified in our MS. Imp-4 was also linked to cell cycle in the GO analysis. The Imp-4 3rd-Z-4% cargoes include the regulator of chromosome condensation RCC1 and the Ser/Thr protein kinase PLK1, and the 2nd-Z-15% cargoes include the sister chromatid cohesion protein PDS5 homolog PDS5B. Imp-4 was also linked to programed cell death or apoptosis in the GO analysis. The representative related 3rd-Z-4% cargoes are the death-promoting transcriptional repressor BCLAF1 and the tumor suppressor ARF (CDKN2A), and the 2nd-Z-15% cargoes are ribosomal L1 domain-containing protein 1 (RSL1D1) and apoptosis- inducing factor 1 (AIFM1).

Imp-5 cargoes Few characteristics are unique to the Imp-5 3rd-Z-4% cargo cohort. However, this cohort includes proteins related to ribosome biogenesis, such as rRNA 2’-O-methyltransferase fibrillarin (FBL), H/ ACA ribonucleoprotein complex subunit 1 (GAR1), and the ribosome biogenesis protein BOP1. This group also includes proteins related to nucleosome or chromatin organization, including spindlin-1 (SPIN1), protein DEK, the methyl-CpG-binding domain protein MBD2, and the paired amphipathic helix protein SIN3B (Supplementary file 5C). SR-rich SFs are also included as described. The Imp-5 2nd-Z-15% cargoes include many ARPs, proteins related to spindle organization or microtubule-

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 18 of 31 Tools and resources Biochemistry Cell Biology based processes, and several CDKs (Supplementary file 5E). Thus, a portion of the Imp-5 cargoes may be involved in cytokinesis. A number of translation initiation factors (eIFs) and elongation factors, many of which are annotated with nuclear localization (Supplementary file 1), are also among the Imp-5 2nd-Z-15% cargoes.

Imp-7 and -8 cargoes The cognate NTRs Imp-7 and -8 share many 3rd-Z-4% and 2nd-Z-15% cargoes (Figure 4; Supplementary file 5A). The major cargoes of these NTRs are a range of mRNA SFs, but by the third Z-scores, snRNPs were identified only as Imp-7 and not Im-8 cargoes (Supplementary file 5C). Additional divergences can be observed between the Imp-7 and -8 cargoes (Supplementary file 5C and 5E). HMG proteins were identified only as Imp-7 3rd-Z-4% and 2nd-Z-15% cargoes, whereas more RPs were identified as Imp-8 cargoes. Proteins related to cell cycle regulation, the mitotic checkpoint protein BUB3, cell division cycle 5-like protein (CDC5L), and CDK12, are included in the Imp-7 3rd-Z-4% cargoes, and the Ser/Thr protein kinase PLK1 is a 2nd-Z-15% cargo, but these proteins are not Imp-8 cargoes. Many eIFs are Imp-8 but not Imp-7 2nd-Z-15% cargoes.

Imp-9 cargoes The Imp-9 cargoes include many RPs and mRNA SFs. Proteins that are important for DNA packaging or nucleosome organization were also identified as Imp-9 cargoes (Supplementary file 5C and 5E). Histone H2A.Z, which is located in specific regions on chromosome (Weber and Henikoff, 2014), is ranked first and third in the third and second Z-scores, respectively. Additionally, the linker histone H1 (H1F0), histone-lysine N-methyltransferase 2A (KMT2A), and the SPT16 and SSRP1 subunits of the FACT complex, which regulates histone H2A.Z (Jeronimo et al., 2015), were also identified as 3rd-Z-4% cargoes. Among the Imp-9 2nd-Z-15% cargoes, other histones, DNA topoisomerase I (TOP1) and IIa (TOP2A), HMG proteins, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1 (SMARCE1), and scaffold attachment factor B1 (SAFB) are included.

Imp-11 cargoes Imp-11 was linked to developmental processes in the GO analysis (Figure 5; Supplementary file 6A), and few proteins with typical nuclear functions, such as DNA replication, nucleosome organization, and transcription, were found among the Imp-11 3rd-Z-4% cargoes (Supplementary file 5C). The Imp-11 2nd-Z-15% cargoes include several proteins related to nuclear division, such as Pogo transposable element with ZNF domain (POGZ), a-endosulfine (ENSA), CDK regulatory subunit 2 (CKS2), and the Ser/Thr protein kinase NEK7 (Supplementary file 5E). Many ARPs, tubulins and their related factors, tRNA ligases, and mRNA SFs are also in the Imp-11 2nd-Z-15% cargoes.

Exp-4 cargoes The subunits of RNAP II elongation factors and mRNA processing factors are the representative Exp-4 cargoes, although they are also cargoes of several other NTRs (Supplementary file 5C and 5E). PAF1C subunit parafibromin (CDC73) is an Exp-4 3rd-Z-4% cargo, and other PAF1C subunits, that is, PAF1 and RTF1, FACT complex subunits, i.e., SSRP1 and SPT16 (SUPT16H), and elongation complex protein 2 (ELP2) are 2nd-Z-15% cargoes. A variety of mRNA processing factors, including 3’-end processing factors and THO complex subunits, are also Exp-4 3rd-Z-4% and 2nd-Z-15% cargoes. Thus, the factors that act in processes from transcription elongation to mRNA export are included in the Exp-4 cargoes. As discussed for another bi-directional NTR Imp-13, the possibility cannot be denied that the identified Exp-4 candidate cargoes include export cargoes.

Seemingly non-nuclear proteins A number of nucleoporins (NUPs), which are the components of the NPC, were identified as cargoes. Increasing evidence demonstrates that the import of NUPs through NPCs is important for gene expression (Burns and Wente, 2014). Moreover, many mitochondrial proteins are highly ranked. These proteins preferentially localize to the mitochondria due to chaperon-regulated or cotranslational mechanisms in vivo and might interact with NTRs in the in vitro transport system. The transport system contains cytosolic extract and unlabeled (light) mitochondrial proteins in it could be

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 19 of 31 Tools and resources Biochemistry Cell Biology

imported if they interact with NTRs. The (L/H+NTR)/(L/HCtl) values of them can be calculated, because LC-MS/MS can quantify low levels of labeled (heavy) proteins whether they are endogenous to the

recipient nuclei or residual after washing. Thus, mitochondrial proteins with high (L/H+NTR)/(L/HCtl) values are imported proteins even if the import is fortuitous. Nuclear localization is annotated to many mitochondrial proteins (Supplementary file 1), and actual nuclear localization is possible as in the cases of AIFM1 and ATFS-1 (Nargund et al., 2012; Susin et al., 1999). As was the case with the high-throughput cargo identification of the export receptor Exp-1 (CRM1) (Kırlı et al., 2015), our method identified other seemingly cytoplasmic proteins as cargoes. We did not detect direct binding between the NTRs and some of these cytoplasmic proteins, for example, Ras-related Rab family proteins and S100 proteins, in the bead halo assays (Supplementary file 2), but nuclear import by indirect binding is still possible.

Additional remarks Here, we have presented the first complete picture of nuclear import via the 12 importin pathways. The 12 pathways must serve distinct roles because the NTRs are linked to different cellular processes by their cargoes. However, the cargoes are intricately allocated to the NTRs, and each NTR is linked to multiple cellular processes. The biological functions of NTRs designated in this work should be further clarified in future experiments. We used HeLa nuclear extract as the cargo source, but it might not reconstitute all NTR–cargo interactions precisely because proteins in the nuclear extract might have different modifications or binding partners from those in cytoplasm where NTRs bind to cargoes in vivo. Some reported cargoes were ranked lower in the 2nd- and 3rd-Z-ranking, and it might be attributable to these differ- ences of protein states. Alternatively, the transport capacity of our in vitro transport system might not be enough to identify all the cargoes, especially those with low transport efficiency. To reach a definitive conclusion, experiments in vivo might be needed. We could not find any novel motifs that may serve as NTR-binding sites on the identified cargoes using the ungapped motif search method of MEME (Bailey and Elkan, 1994). A more extensive search for such motifs and higher order structures using alternative methods is currently underway.

Materials and methods SILAC-Tp SILAC-Tp has previously been described in detail (Kimura et al., 2014), but we provide a brief description here. HeLa-S3 cytosolic and nuclear extracts were depleted of Imp-b family NTRs with phenyl-Sepharose (GE healthcare), and the nuclear extract was subsequently depleted of RCC1 with a Ran-affinity method and concentrated. The extracts were dialyzed against transport buffer (TB, 20 mM HEPES–KOH (pH 7.3), 110 mM KOAc, 2 mM MgOAc, 5 mM NaOAc, 0.5 mM EGTA, 2 mM DTT, and 1 mg/mL each of aprotinin, pepstatin A, and leupeptin). Adherent HeLa-S3 cells were labeled 13 13 with u- C6 Lys and u- C6 Arg by SILAC (Ong et al., 2002) and seeded onto a glass plate. After rinsing in ice cold TB, the cells were permeabilized with 40 mg/mL digitonin in TB for 5 min on ice and then rinsed again. The permeabilized cells were pretreated with 4 mM RanGDP and an ATP regeneration system in TB for 20 min at 30˚C to remove the residual Imp-b family NTRs and then rinsed. The cells were incubated in transport mixture (50% cytosolic extract, 10% nuclear extract, 1 mM p10/NTF2, and ATP regeneration system in TB) with (+NTR) or without (Ctl) 0.3–0.7 mM of one NTR for 20 min at 30˚C for the import reaction. (The NTR concentrations were optimized using the recombinant cargoes presented in Figure 1—figure supplement 1C.) After rinsing, the cells were incubated in extract mixture (50% cytosolic extract and ATP regeneration system in TB) for 20 min at 30˚C and rinsed with NaCl-TB (TB containing 110 mM NaCl instead of KOAc) to remove the nonspe- cifically binding proteins. To extract the proteins, the cells were suspended in nuclear buffer (20 mM

Tris–HCl, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 2 mM DTT, and 1 mg/mL each of aprotinin, pepstatin A, and leupeptin), sonicated, and centrifuged. Actually, the transport reactions for two NTRs were simultaneously performed with one control reaction and triplicated. The simultaneously processed NTRs were Imp-b and Imp-13, Trn-1 and -2, Imp-7 and -8, Imp-9 and -11, and Imp-5 and Trn-SR, and the reactions for Imp-4 and Exp-4 were performed individually with controls.

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 20 of 31 Tools and resources Biochemistry Cell Biology Peptide analysis by LC-MS/MS After the in vitro transport reaction, 25 mg each of the extracted proteins was concentrated by ace- tone precipitation, reduced with DTT, and alkylated with iodoacetamide. The proteins were digested with trypsin and Lys-C endopeptidase (enzyme/substrate » 1/50) for 16 hr at 37˚C. The peptides were evaporated to dryness, dissolved in Solvent-1 (0.1% TFA and 15% CH3CN), and fractionated on Empore Cation Exchange-SR (3M, Maplewood, Minnesota). For the fractionation, the support was stacked manually inside the tapered end of a micropipette tip, the tip was fixed into the punched lid of a microtube, and the liquids were run by centrifugation (Wis´niewski et al., 2009). The resin was sequentially washed by ethanol and Solvent-1 containing 500 mM ammonium acetate and equilibrated with Solvent-1, and the peptides were then applied. After washing in Solvent-1, the peptides were eluted stepwise by Solvent-1 containing 125, 250, and 500 mM ammonium acetate

and Solvent-2 (5% NH4OH, 30% methanol, and 15% CH3CN). The eluates were evaporated to dry-

ness, and the peptides were dissolved in 0.1% TFA and 2% CH3CN. The peptides were applied to a liquid chromatograph (LC) (EASY-nLC 1000; Thermo Fisher Scien- tific, Waltham, Massachusetts) coupled to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) with a nanospray ion source in positive mode. The LC was performed on a NANO-HPLC capillary column C18 (75 mm x 150 mm, 3 mm particle size, Nikkyo Technos, Tokyo) at 45˚C. The peptides were eluted with a 100-min 0–30% CH3CN gradient and a subsequent 20-min 30–65% gradient in the presence of 0.1% formic acid at a flow rate of 300 nL/min. The Q Exactive-MS was operated in the top-10 data-dependent scan mode. The parameters for the Q Exactive operation were as follows: spray voltage, 2.3 kV; capillary temperature, 275˚C; mass range (m/z), 350–1800; and normalized collision energy, 28%. The raw data were acquired with Xcalibur (RRID:SCR_014593; ver. 2.2 SP1).

Protein identification and quantitation The MS and MS/MS data were searched against the Swiss-Prot database (2014_07–2016_01) using Proteome Discoverer (RRID:SCR_014477; ver. 1.4, Thermo Fisher Scientific) with the MASCOT search engine software (RRID:SCR_014322; ver. 2.4.1, Matrix Science, London). The search parameters were as follows: taxonomy, Homo sapiens; enzyme, trypsin; static modifications, carbamido- methyl (Cys); dynamic modifications, oxidation (Met); precursor mass tolerance, ±6 ppm; fragment mass tolerance, ±20 mDa; maximum missed cleavages, 1; and quantitation, SILAC (R6, K6). The proteins were considered identified when their false discovery rates were less than 5%. The SILAC L/H ratios were also calculated by Proteome Discoverer (ver. 1.4) with the default setting: show the raw quan values, false; minimum quan value threshold, 0; replace missing quan values with minimum intensity, false; use single-peak quan channels, false; apply quan value corrections, true; reject all quan values if not all quan channels are present, false; fold change threshold for up-/down-regulation, 1.5; maximum allowed fold change, 100; use ratios above maximum allowed fold change for quantification, false; percent co-isolation excluding peptides from quantification, 100; protein quantification, use only unique peptides; experimental bias, none. Proteins with L/H count 1 were included in further analysis. The L/H counts are shown in Supplementary file 1. To access the mass spectrometry data, see below.

From the SILAC quantitation values of the control and +NTR reactions, the +NTR/Ctl = (L/H+NTR)/

(L/HCtl) ratio of each protein was calculated, and the Z-score of the log2(+NTR/Ctl) of each protein was calculated within each replicate. Z À score ¼ ðX À Þ=s

where X is log2ðþNTR=CtlÞ ¼ log2½ðL=HþNTRÞ=ðL=HCtlÞ of each protein, is the mean of X, and s is the standard deviation of X.

Reported cargo rate and recall To calculate reported cargo rate (a lower bound on precision) and recall (sensitivity), we used the 27 and 25 reported cargoes of Trn-1 as the positive examples of the 2nd- and 3rd-Z-rankings, respectively. We do not have explicit labeling of negative examples. Most likely some portion of the proteins not reported as cargoes are genuine cargoes, but it is difficult to estimate that portion. Therefore, as a rough guide we tallied statistics under two simple assumptions: (i) that all proteins

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 21 of 31 Tools and resources Biochemistry Cell Biology not reported as cargoes should be treated as negative examples and (ii) that in the proteins not reported as cargoes, proteins annotated in Uniprot (RRID:SCR_002380) as having non-nuclear subcellular localization should be treated as negative examples and the other proteins excluded from the analysis (treated as neither positive nor negative). The first definition yielded 1622 and 1210 negative examples in the 2nd- and 3rd-Z-ranking, respectively, and the second definition 259 and 178 in the 2nd- and 3rd-Z-ranking, respectively. Since the first definition is maximally pessimistic, it allows estimation of an upper bound on the rate of false positives, while the second definition is more optimistic. Reported cargorateðiÞ ¼ pðiÞ=½pðiÞ þ nðiÞ

RecallðiÞ ¼ pðiÞ=P

where pðiÞ denotes the number of previously reported cargoes (a lower bound on the number of positive examples) and nðiÞ denotes the number of negative examples in the top i%; while P denotes the total number of previously reported cargoes.

Gene ontology analysis GO (RRID:SCR_002811) analyses were performed using g:Profiler (RRID:SCR_006809; r1488- 1536_e83_eg30) (Reimand et al., 2016). The search parameters were the following: organism, Homo sapiens; significance threshold, g:SCS; statistical domain size, all known genes; GO version, GO direct 2015-12-09 to 2016-01-21, releases/2015-12-08.

Phylogenetic analysis of the 12 Imp-b family NTRs The phylogeny was inferred by maximum likelihood using RAxML (RRID:SCR_006086; ver. 8.1.17) (Stamatakis, 2006) with 1000 bootstrap replicates and the LG model with gamma-distributed rate variation. The amino acid sequences were aligned using Clustal Omega (RRID:SCR_001591; ver. 1.2.0) (Sievers et al., 2011) with the default parameters, and the resulting multiple alignments were trimmed using trimAl (ver. 1.2) (Capella-Gutie´rrez et al., 2009) in gappyout mode.

Hierarchical clustering of the 11 Imp-b family NTRs based on the degree of overlap of the 3rd-Z-4% cargoes We performed a hierarchical clustering of the Imp-b family NTRs based on their cargo profile similarities using Ward’s method with Euclidean distance as implemented in the software R (RRID:SCR_ 001905; R Development Core Team, 2012). Here, we omitted Imp-b because its cargoes include many Imp-a-dependent indirect cargoes. To define a cargo profile for each NTR, we first defined a set of cargoes by merging the 3rd-Z-4% cargoes of the 11 NTRs other than Imp-b, which yielded a total of 426 cargoes. We then defined length 426 binary vectors for each NTR with a 1 for each cargo in the top 4% list and a 0 otherwise and input these 11 vectors into R to perform the clustering.

Bead halo assay The proteins and Escherichia coli extracts were prepared as described (Kimura et al., 2013a). The bead halo assays (Supplementary file 2) were performed as described (Patel and Rexach, 2008). Briefly, GST or GST-NTR was immobilized on glutathione-Sepharose (GE healthcare), and mixed with an extract of E. coli expressing a GFP-fusion protein in EHBN buffer (10 mM EDTA, 0.5% 1,6- hexanediol, 10 mg/mL bovine serum albumin, and 125 mM NaCl), and the binding was observed by fluorescent microscopy. The GTP-fixed mutant of Ran Q69L-Ran, which inhibits specific NTR–cargo interactions, was added to determine the specificity of the binding. The expression and degradation levels of the GFP-fusion proteins were analyzed, and the concentrations of GFP-moieties were quantified by triplicate quantitative Western blotting of the extracts with an anti-GFP antibody. Because the GFP-moiety weakly bound to GST-Trn-1, GST-Trn-2, and GST-Trn-SR in the bead halo assay, the concentrations of the GFP-fusion proteins and GFP (control) were equalized, and images were acquired and processed under identical condition. In contrast, because the GFP-moiety does not bind to GST-Imp-13, GST-Imp-b, or GST-Imp-a, the control reaction mixture for these NTRs contained higher concentration of GFP than any other GFP-fusion proteins. Three images (GST, GST-

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 22 of 31 Tools and resources Biochemistry Cell Biology NTR, and GST-NTR + Q69L-Ran) for each GFP-fusion protein were acquired under identical conditions, and the background intensities and dynamic ranges were equalized.

Cell line HeLa-S3 (RRID:CVCL_0058; mycoplasma, not detected) was obtained from Dr. Fumio Hanaoka (RIKEN).

Antibodies See Supplementary file 12B.

GFP-fusion proteins used for in vitro transport The GFP-fusion proteins used in Figure 1—figure supplement 1C were prepared as described (Kimura et al., 2013a). For accessions and references, see Supplementary file 12B. The SOX2 cDNA (pF1KB9652) was from Kazusa DNA Res. Inst. (Kisarazu, Japan), and the others were cloned from a HeLa cDNA library (SuperScript, Life Technology) by PCR.

Database deposition The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (RRID:SCR_004055; http://www.proteomexchange.org/) via the PRIDE (RRID:SCR_003411; Vizcaı´no et al., 2016) partner repository with the dataset identifier PXD004655. The .msf and .raw data files of each experiment summarized in Supplementary file 1 are listed in Supplementary file 12A. The protein and peptide quantitation results can be seen by opening .msf files by Proteome Discoverer software. To see spectra and chromatograms, .msf files and corresponding .raw files must be in the same local directory. A demo version of Proteome Discoverer can be downloaded at the Thermo Scientific omics software portal site (https://portal.thermo-brims. com/).

Acknowledgements We thank Martin Beck, Alessandro Ori, and Kentaro Tomii for helpful discussion, Masahiro Oka for a supporting experiment, Shoko Motohashi and Ai Watanabe for technical assistance, and Hiroshi Mamada for a plasmid. We also thank the Support Unit for Bio-Material Analysis, RIKEN BSI Research Resource Center, especially Kaori Otsuki, Masaya Usui, and Aya Abe for MS analysis. This work was supported by JSPS KAKENHI Grant Number 15K07064 and a RIKEN FY2014 Incentive Research Project to MK, KAKENHI Grant Numbers 26251021, 26116526, and 15H05929 to NI, and the Platform Project for Supporting in Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Japan Agency for Medical Research and Development (AMED) to KI.

Additional information

Funding Funder Grant reference number Author Japan Society for the Promo- KAKENHI,15K07064 Makoto Kimura tion of Science RIKEN FY2014 Incentive Research Makoto Kimura Project Japan Agency for Medical Re- Platform Project for Kenichiro Imai search and Development Supporting in Drug Discovery and Life Science Research Japan Society for the Promo- KAKENHI,26251021 Naoko Imamoto tion of Science Japan Society for the Promo- KAKENHI,26116526 Naoko Imamoto tion of Science

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 23 of 31 Tools and resources Biochemistry Cell Biology

Japan Society for the Promo- KAKENHI,15H05929 Naoko Imamoto tion of Science

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Author contributions MK, Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Methodol- ogy, Writing—original draft, Project administration, Writing—review and editing; YM, Formal analysis, Investigation, Visualization, Writing—review and editing; KI, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing—review and editing; SK, Conceptualization, Resources, Methodology, Writing—review and editing; PH, Supervision, Validation, Investigation, Methodology, Writing—review and editing; NI, Conceptualization, Supervision, Funding acquisition, Validation, Project administration, Writing—review and editing

Author ORCIDs Makoto Kimura, http://orcid.org/0000-0003-0868-5334 Naoko Imamoto, http://orcid.org/0000-0002-2886-3022

Additional files Supplementary files . Supplementary file 1. Results of SILAC-Tp. Each sheet contains the result of SILAC-Tp with one of

the 12 NTRs. The proteins that exhibited þNTR=Ctl ¼ ðL=HþNTRÞ=ðL=HCtlÞ values at least once in the three replicates (Experiments 1–3) are listed with the log2½ðL=HþNTRÞ=ðL=HCtlÞ values. The second and third Z-scores and the ranks according to those scores are also presented. The 2nd-Z-15% and 3rd-Z-4% cargoes are indicated in cyan. The LC-MS/MS quantitation data for each replicate (Experi- ments 1–3) are also included. Light/Heavy, the median of quantified L/H values; Light/Heavy count, the number of quantified values; Light/Heavy variability, coefficient-of-variation for log-normal distributed data. aReport: The proteins listed by Chook and Su¨el (2011) are regarded as reported cargoes, and the references are provided in the Legend sheet. For Imp-b, only direct cargoes are listed. For Trn-2, the reported Trn-1 cargoes are listed. bDirect Binding: The results of the bead halo assays (Supplementary file 2) are summarized. ++ or +, positive; ± or –, negative. cGO Nucleus: Annotated with ‘nucleus’ in Gene Ontology (term type, cellular component). The rows can be sorted into preferable orders with Excel. To access the mass spectra, chromatograms, or raw data, see Supplementary file 12A. Statistics: For each experiment, the number of proteins assigned with

log2½ðL=HþNTRÞ=ðL=HCtlÞ values (proteins assigned with Z-scores), the mean and standard deviation (S.D.) of log2½ðL=HþNTRÞ=ðL=HCtlÞ are listed. Z À score ¼ ðX À Þ=s where X ¼ log2½ðL=HþNTRÞ=ðL=HCtlÞ of each protein, is the mean of X in one experiment, and s is the S.D. of X . DOI: 10.7554/eLife.21184.017 . Supplementary file 2. NTR–cargo direct binding. The direct binding of the candidate cargoes to the NTRs was analyzed by bead halo assay. From well-characterized proteins that have not been reported as cargoes, (i) proteins ranked high (within the top 15% in the 2nd-Z-rankings or 4% in the 3rd-Z-rankings), around presumptive cutoffs (within about top 15–25% in the 2nd-Z-rankings), or lower and (ii) highly ranked proteins that are suspected as indirect cargoes or false positives based on their well-known features, e.g., PMPCA, GALE, UAP1, NQO2, EEF1A2, RAB2A, RAB8A, S100A4, S100A6, S100A13, and S100P, were selected and analyzed. Proteins in (i) verify the cargo identification and cutoff setting, and proteins in (ii) serve for finding indirect cargoes and false positives. The negative rate of these bead halo assays should be higher than the true overall false positive rate of the SILAC-Tp, because proteins in (ii) were selected preferentially. GST or GST-NTR was attached to glutathione-Sepharose beads, mixed with an extract of E. coli expressing GFP or a GFP-fusion protein, and observed by fluorescence microscopy. Q69L-Ran, which inhibits the NTR–cargo functional binding, was added as appropriate. The contrast of the bead fluorescence between the GST and GST-NTR indicates the binding, and the inhibition of this binding by Q69L-Ran certifies the specificity of the binding; ++ or +, positive; ± or –, negative. Summary of the results (p2–5): The results are

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 24 of 31 Tools and resources Biochemistry Cell Biology summarized in both 2nd- and 3rd-Z-rank order. The 2nd-Z-15% and 3rd-Z-4% cargoes are indicated by cyan, and positive binding (++ or +) is indicated by blue. Trn-1 (p6–8): The GFP-fusion proteins were divided into five groups (A–E) according to the expression levels. Because GFP binds weakly to Trn-1, the concentrations of GFP (control) and GFP-fusion proteins were equalized within each group, and the binding was observed in the same conditions. The images are comparable within a group. Trn-2 (p9): GFP weakly binds to Trn-2, and the concentrations of GFP and GFP-fusion proteins were equalized. The images are comparable. Proteins whose ranks differed substantially between the Trn-1 and Trn-2 Z-ranking were assayed. Imp-13 (p10–13): GFP does not bind to Imp- 13, and GFP was added to the control mixture at the highest concentration. Three images (GST, GST-Imp-13, and GST-Imp-13 + Q69L-Ran) for each GFP-fusion protein were acquired under identical conditions, and the background intensities and dynamic ranges were equalized. Trn-SR (p14–16): GFP weakly binds to Trn-SR, and the procedures were similar to those used for Trn-1. The GFP- fusion proteins were divided into four groups (A–D), and the images are comparable within a group. Imp-a/b (p17–20): GFP does not bind to Imp-a or -b, and the procedures were similar to those used

for Imp-13. GST-Imp-a2 lacks the N-terminal Imp-b-binding domain. Western blotting (p21–22): The GFP-fusion proteins in the E. coli extracts were relatively quantified by Western blotting using an anti-GFP antibody (Roche). The extracts containing the amounts of protein (ng) indicated at the bot- toms were loaded. The arrowheads indicate the expected full-length products. The GFP-moieties including those of the partial products were quantified by chemiluminescence. GFP was used as the standard. The Western blots were replicated more than three times. Accessions and sequences (p23–27): The cDNAs were cloned from a HeLa cDNA library by PCR. The accession numbers of the proteins are listed. If the sequence of a used protein is different from that in the database, the deleted, substituting, or inserted amino acids are indicated by the colors. The sequences that matched perfectly are not presented. DOI: 10.7554/eLife.21184.018 . Supplementary file 3. The 2nd-Z-15% cargoes of the 12 NTRs. The 2nd-Z-15% cargoes of each NTR are listed by the gene names in the 2nd-Z-rank orders. The ranks by the third Z-scores are also shown. Cyan in the rank columns indicates the 2nd-Z-15% and 3rd-Z-4% cargoes. Colors in the gene name columns: magenta, reported cargoes; blue, cargoes bound directly to the NTR in the bead halo assays (Supplementary file 2); light blue, cargoes bound directly to Imp-a but not Imp-b; gray, proteins that did not bind to the NTRs; yellow, Imp-a; and green, reported export cargoes. For the 3rd-Z-4% cargoes, see Figure 3. DOI: 10.7554/eLife.21184.019 . Supplementary file 4. Example of extracted ion chromatograms (EICs) of peptides. EICs of GLUD1 peptides in the SILAC-Tp with Trn-1: As a general problem of high-throughput LC-MS/MS quantitation, quantitative values of proteins with fewer quantified peptides deviate among the replicates. Referring to EICs of the quantified peptides is useful to avoid misidentification of cargoes. For example, the L/H ratios of a Trn-1 2nd-Z-15% cargo GLUD1 (P00367, ranked 110th and 342nd by the second and third Z-score, respectively) deviated largely in the three replicates of SILAC-Tp with Trn-1 (Supplementary file 1). Panels (A–H) show EICs of the indicated peptide (trypsin targets, K and R, are written in lower cases) in the three (three Ctl and there +Trn) experiments (some peptides were not identified in all the experiments). Magenta letters indicate the quantified peptides and L/H rations. In panel (A), the elution time of the peptide TAMkYNLGLDLr differs largely between the expriment-1 Ctl and experiment-2 +Trn-1, the peak shape of the experiment-2 +Trn-1 is irregular, and the L/H ratio of it is much higher than those of other peptides in +Trn-1 experiments (B and E). Thus, there is concern about misidentification. Because the L/H count of GLUD1 in the experiment-2 +Trn-1 is two (TAMkYNLGLDLr and NLNHVSYGr, A and B) and the L/H ratio of a protein is defined as the median, the L/H ratio of GLUD1 in the expriment-2 +Trn-1 is affected by the L/H ratio of TAMkYNLGLDLr. Exclusion of the L/H ratio of TAMkYNLGLDLr in the experiment-2 +Trn-1 lowers the Z-score rank of GLUD1 significantly. In panel (C), the chromatogram of the peptide HGGTIPIVP- TAEFQDr in the experiment-1 Ctl has an irregular peak, and the L/H ratio of it is much higher than those of other peptides in the Ctl experiments (A–H). Thus, overlap with other peptide or other fail- ures may be possible. However, the L/H count of GLUD1 in the experiment-1 Ctl is four (TAM- kYNLGLDLr, HGGTIPIVPTAEFQDr, ALASLMTYk, and GASIVEDkLVEDLr) and the value of HGGTIPIVPTAEFQDr does not affect the median. (The L/H ratio of TAMkYNLGLDLr, whose EIC

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 25 of 31 Tools and resources Biochemistry Cell Biology differ between the experiment-1 Ctl and expriment-2 +Trn in (A) as mentioned above, may affect the L/H ratio of GLUD1 in the experiment-1 Ctl, but we assumed that it is reliable.) As above, the L/ H ratios of proteins with low L/H counts (Supplementary file 1) may be affected by LC-MS/MS arti- facts, and misidentification can be avoided by referring to the EICs. All the EICs and MS spectra in this work can be accessed by downloading the mass spectrometry data and Proteome Discoverer software (see the Materials and methods and Supplementary file 12A). DOI: 10.7554/eLife.21184.020 . Supplementary file 5. Redundancy of NTRs: Cargoes shared by NTRs. (A) The numbers of the 2nd- Z-15% cargoes shared by two NTRs. For the 3rd-Z-4% cargoes, see Figure 4. (B) Redundancy of the 3rd-Z-4% cargoes. Many proteins are included in the 3rd-Z-4% cargoes of multiple NTRs. The ranks of these cargoes in the 3rd-Z-rankings for all 12 NTRs are presented. (C) Relationships between the NTRs and the characteristics of their 3rd-Z-4% cargo proteins. The 3rd-Z-4% cargoes are grouped according to their characteristics (functions or biological processes that the proteins act in), and their ranks are presented as in (B). To make our points clear, typical terms for the protein characteristics and typical proteins related to the terms have been selected with reference to Gene Ontology (GO) and UniProt. Thus, the terms in this sheet are slightly different from those in the databases, fewer proteins than annotated in the databases are grouped, and the list is redundant. For the complete linkages between the GO terms and the 3rd-Z-4% cargoes, see Supplementary file 7. (D) Redun- dancy of the 2nd-Z-15% cargoes. The ranks of the 2nd-Z-15% cargoes are presented as in (B). (E) Relationships between the NTRs and the characteristics of their 2rd-Z-15% cargo proteins. The 2nd- Z-15% cargoes are grouped, and their ranks are presented in a manner similar to that in (C). For the complete linkages between the GO terms and the 2nd-Z-15% cargoes, see Supplementary file 8. DOI: 10.7554/eLife.21184.021 . Supplementary file 6. GO term enrichments of the identified cargoes. (A) Extraction of the GO term enrichments of the 2nd-Z-15% cargoes. The 2nd-Z-15% cargoes were analyzed for GO term enrichment in (C). The terms that were significantly enriched (p<0.05, cyan) in the 2nd-Z-15% cargoes of four or fewer NTRs were selected, and terms that represent many similar terms are presented. With the p-values, the numbers (#) of cargoes annotated with each of the terms are presented. Total No. represents the number of proteins annotated with each term in the database. Related terms are bundled in the same color. For the 3rd-Z-4% cargoes, see Figures 5 and 6. The correspondences between each 2nd-Z-15% cargo and GO term are summarized in Supplementary file 10. All the GO terms annotated to the 2nd-Z-15% cargoes are listed in Supplementary file 8. (B) Full table of the GO term enrichments of the 3rd-Z-4% cargoes. The 3rd- Z-4% cargoes were analyzed for GO term enrichment. For all combinations of GO terms and NTRs, the p-values for the term enrichments in the 3rd-Z-4% cargoes and the numbers (#) of cargoes annotated with the terms are presented. The numbers following ‘# in’ are the total numbers of 3rd-Z-4% cargoes. Total No. represents the number of proteins annotated with each term in the database. Cyan, p<0.05. Figures 5 and 6 were extracted from this table. This table was derived from Supplementary file 7, and see Supplementary file 7 to retrieve the protein accessions. (C) Full table of the GO term enrichments of the 2nd-Z-15% cargoes. The 2nd-Z-15% cargoes were analyzed and are presented in a manner similar to that in (B). (A) was extracted from this table. This table was derived from Supplementary file 8, and see Supplementary file 8 to retrieve the protein accessions. DOI: 10.7554/eLife.21184.022 . Supplementary file 7. The 3rd-Z-4% cargoes annotated with GO terms. With respect to each NTR, the accessions of the 3rd-Z-4% cargoes annotated with each GO term are listed. Cyan, significant term enrichment (p<0.05) in the 3rd-Z-4% cargoes of the NTR. Total No. represents the number of proteins annotated with each term in the database. Supplementary files 6B and 9 were derived from this table. For the 2nd-Z-15% cargoes, see Supplementary file 8. DOI: 10.7554/eLife.21184.023 . Supplementary file 8. The 2nd-Z-15% cargoes annotated with GO terms. With respect to each NTR, the accessions of the 2nd-Z-15% cargoes annotated with each GO term are listed. Cyan, significant term enrichment (p<0.05) in the 2nd-Z-15% cargoes of the NTR. Total No. represents the number of proteins annotated with each term in the database. Supplementary files 6C and 10 were derived from this table. For the 3nd-Z-4% cargoes, see Supplementary file 7.

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 26 of 31 Tools and resources Biochemistry Cell Biology DOI: 10.7554/eLife.21184.024 . Supplementary file 9. Correspondences between the 3rd-Z-4% cargoes and GO terms. Each sheet shows the correspondences between the 3rd-Z-4% cargoes of one NTR and selected GO terms. A term annotation to a cargo is indicated by ‘1’ in the corresponding cell. Reported cargoes are indicated by magenta in the gene name cells, and the results of the bead halo assays (Supplementary file 2) are also indicated by colors in the gene name cells: blue, cargoes directly bound to the NTR; light blue, cargoes directly bound to Imp-a but not Imp-b; gray, proteins that did not bind to the NTR. GO terms that represent many similar terms were selected from the terms enriched significantly (p<0.05) for the 3rd-Z-4% cargoes of each NTR, and broadly defined terms were deselected. Magenta and orange in the term ID cells indicate terms that are significantly enriched for the cargoes of four or fewer NTRs, and of them magenta indicates the terms presented in Figures 5 and 6. Related GO terms are bundled in the same color, and different colors are used to distinguish the columns easily. The NTRs added in the transport reactions (white in the rank cells) were not analyzed. This table was derived from Supplementary file 7. For 2nd-Z-15% cargoes, see Supplementary file 10. DOI: 10.7554/eLife.21184.025 . Supplementary file 10. Correspondences between the 2nd-Z-15% cargoes and GO terms. Each sheet shows the correspondences between the 2nd-Z-15% cargoes of one NTR and selected GO terms. A term annotation to a cargo is indicated by ‘1’ in the corresponding cell. Reported cargoes are indicated by magenta in the gene name cells, and the results of the bead halo assays (Supplementary file 2) are also indicated by colors in the gene name cells: blue, cargoes directly bound to the NTR; light blue, cargoes directly bond to Imp-a but not Imp-b; gray, proteins that did not bind to the NTR. GO terms that represent many similar terms were selected from the terms enriched significantly (p<0.05) for the 2nd-Z-15% cargoes of each NTR, and broadly defined terms were deselected. Magenta and orange in the term ID cells indicate terms that are significantly enriched for the cargoes of four or fewer NTRs, and of them magenta indicates the terms presented in Supplementary file 6A. Related GO terms are bundled in the same color, and different colors are used to distinguish the columns easily. The NTRs added in the transport reactions (white in the rank cells) were not analyzed. This table was derived from Supplementary file 8. For 3rd-Z-4% cargoes, see Supplementary file 9. DOI: 10.7554/eLife.21184.026 . Supplementary file 11. mRNA processing factors, ribosomal proteins, and transcription factors. (A) Ranks of mRNA processing factors. All of the 2nd-Z-15% and 3rd-Z-4% cargoes that are annotated with mRNA processing in Gene Ontology (GO) are listed with the ranks in the 2nd- and 3rd-Z-rankings. The color scale is set by percentile rank as indicated. Figure 7 was extracted from this table. (B) The 3rd-Z-rankings of ribosomal proteins. The ranks of the ribosomal proteins in the 3rd-Z-rankings of the 12 NTRs are presented. The color scale is set by percentile rank as indicated. For the 2nd-Z-rankings, see Figure 8. (C) Extracts of the GO term enrichments of the transcription factors found in the 2nd-Z-15% cargoes. Seventeen to 36 proteins in the 2nd-Z-15% cargoes of each NTR are annotated with ‘transcription factor activity, sequence-specific DNA binding’ or ’transcription factor activity, protein binding’ in GO. The factors were analyzed for GO term enrichment (term type, biological process, BP) in (D). Typical terms that represent similar terms were extracted from (D). The p-value for the term enrichment and the number (#) of factors annotated with the term are presented. Total No. represents the number of proteins annotated with each term in the database. Cyan, p<0.05. Related terms are bundled in the same color. (D) Full table of the GO term enrichments of the transcription factors found in the 2nd-Z-15% cargoes. The 2nd-Z-15% cargoes that are annotated with ‘transcription factor activity, sequence-specific DNA binding" or ’transcription factor activity, protein binding’ in GO were analyzed for GO term enrichment (term type, BP). The analyzed transcription factors are listed at the bottom. For all of the combinations of GO terms and NTRs, the p-value for the term enrichment and the number (#) of transcription factors annotated with the term are presented. The numbers following ‘# in’ are the total numbers of transcription factors in the 2nd- Z-15% cargoes. Total No. represents the number of proteins annotated with each term in the database. Cyan, p<0.05. (C) was extracted from this table. (E) SRSRSR motif in the 3rd-Z-4% cargoes. The 3rd-Z-4% cargoes that contain an ‘SRSRSR’ hexa-peptide sequence were counted. DOI: 10.7554/eLife.21184.027

Kimura et al. eLife 2017;6:e21184. DOI: 10.7554/eLife.21184 27 of 31 Tools and resources Biochemistry Cell Biology . Supplementary file 12. MS data files, recombinant cargoes, and antibodies. (A) MS data files. The mass spectrometry proteomics data (.msf and .raw files) have been deposited to the ProteomeX- change Consortium (http://www.proteomexchange.org/) with the dataset identifier PXD004655. The results of protein and peptide identification and quantitation are summarized in Supplementary file 1, and the .msf and .raw data files corresponding to each experiment in Supplementary file 1 are listed in this table. The quantitation results can be seen by opening .msf files by Proteome Discov- erer software. To see spectra and chromatograms, .msf files and corresponding .raw files must be in the same local directory. A demo version of Proteome Discoverer can be downloaded at the Thermo Scientific omics software portal site (https://portal.thermo-brims.com/). (B) GFP-fusion proteins used for in vitro transport and antibodies. DOI: 10.7554/eLife.21184.028

Major datasets The following dataset was generated:

Database, license, and accessibility Author(s) Year Dataset title Dataset URL information Kimura M, Imamoto 2016 SILAC-Tp (12 importins) http://www.ebi.ac.uk/ Publicly available at N pride/archive/projects/ the Pride Archive PXD004655 (accession no: PXD004655)

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